Fibroblast growth factor

ABSTRACT

The invention relates to compositions and methods for inhibiting cell proliferation, especially angiogenesis. The invention specifically relates to fusions of the extracellular domain of a fibroblast growth factor receptor (FGFR) with a heterologous oligomerization domain, such as that contained in an immunoglobulin, to provide potent FGFR antagonists.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of U.S. application Ser. No.09/499,846, filed Feb. 7, 2000, which claims the benefit of U.S.Provisional Application No. 60/119,002, filed Feb. 8, 1999; which arehereby incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

[0002] The invention relates to compositions and methods for inhibitingcell proliferation, especially angiogenesis. The invention specificallyrelates to fusions of the extracellular domain of a fibroblast growthfactor receptor (FGFR) with an immunoglobulin (Ig), especially an Ig Fcregion, as an FGFR antagonist. The invention also relates to novelFGFR-Ig fusion proteins.

BACKGROUND OF THE INVENTION

[0003] Angiogenesis, or development of new blood vessels, is implicatedin a host of diseases including tumorigenesis, metastasis and tumorgrowth, retinopathies, neovascular ocular disorders, and postangioplastyor postatherectomy restenosis (Bicknell et al. (1996) Curr. Opin. Oncol.8:60-65; Gariano et al. (1996) Survey Ophthalmol. 40:481-490; and Wilcox(1993) Am. J. Cardiol. 72:88E-95E).

[0004] Expression and secretion of angiogenic factors by tumors has beeninvestigated. It has been suggested that because tumors express multipleangiogenic factors, broad spectrum antagonists of angiogenesis canprovide effective means of tumor stabilization. Anti-angiogenicapproaches to tumor therapy have been defined to involve interferencewith growth, migration, and differentiation of blood vessels associatedwith tumor growth. Anti-angiogenic agents have been categorized toinclude protease inhibitors, modulators of cytokines, heparin-likemolecules, and antagonists of vascular growth factors. Growth factorantagonists have been categorized to include heparin-like molecules,angiogenin antagonists, antisense fibroblast growth factor, DS 4152,suramin analogs, and protein-bound saccharide-K (Bicknell et al. (1996)Curr. Opin. Oncol. 8:60-65).

[0005] Various growth factors and growth factor receptors are known tobe associated with particular types of tumors. At the molecular level,growth factors and growth factor receptors belong to multi-memberfamilies categorized based on structural and functional characteristics.Fibroblast growth factor (FGF) is involved in growth and differentiationof a number of cell types, and can contribute significantly totumorigenicity. The FGF family includes FGF-1 or acidic FGF (aFGF),FGF-2 or basic FGF (bFGF), FGF-7 or KGF, oncogene products FGF-3 orint-2, hsp/Kaposi-FGF (K-FGF or FGF-4), FGF-5, and FGF-6. These membersof the FGF family bind heparin, may exhibit mitogenic activity towardvarious cells, and may be potent mediators of angiogenesis (Pontalianoet al. (1994) Biochemistry 33:10229-10248; Kiefer et al. (1991) GrowthFactors 5:115-127).

[0006] FGF receptor (FGFR) includes FGFR1 or flg, FGFR2 or bek, FGFR3 orcek2, and FGFR4 (Kiefer et al. (1991) Growth Factors 5:115-127). FGFRbelongs to the tyrosine kinase family of receptors and to theimmunoglobulin (Ig) supergene family. mRNA splicing variants of FGFRexist that produce secreted and transmembrane forms of the receptorswith various ligand binding affinities and specificities. Intransmembrane forms of the receptor, the tyrosine kinase domain isintracellular and the (Ig)-like domains are extracellular. Bothtransmembrane and secreted forms bind FGF. Heparin and related compoundspromote the interaction between FGF and FGFR by acting as cofactors indimerization or higher oligomerization of FGFR. The dimerization processis thought to be necessary for activation of FGFR.

[0007] FGFR fusion proteins present the possibility of constructingpreoligomerized, particularly predimerized forms of FGFR. Suchpreoligomerized forms would be useful as potent and therapeuticallyeffective inhibitors of FGF-mediated cell proliferation. FGFRantagonists would be especially useful to treat diseases involvingangiogenesis.

[0008] Monomeric forms of the FGFR extracellular domain have been usedto inhibit FGF-mediated events (Kiefer et al. (1991) Growth Factors5:115-127). However, preoligomerized forms of the FGFR extracellulardomain have not been used as FGFR antagonists. Thus, there is a need forproviding preoligomerized forms of FGFR extracellular domain asantagonists of FGFR. Given the implicated role of this ligand/receptorsystem in angiogenesis, and the breadth of involvement of angiogenesisin several malignancies and other disorders, the approach promises auseful tool in providing an effective therapy for such disorders.

[0009] Receptor-immunoglobulin (Ig) fusion proteins have been used inthe art. For example, an Ig fusion protein with a human tumor necrosisfactor receptor has been applied to treatment of rheumatoid arthritisand septic shock (Moreland et al. (1997) New Engl. J. Med. 337:141-147;Fisher et al. (1996) New Engl. J. Med. 334:1697-1702). An Ig fusionprotein with urokinase plasminogen activator (uPA) has been used as auPA receptor antagonist to inhibit angiogenesis and tumor growth (Min etal. (1996) Cancer Res. 56:2428-2433). WO 95/21258 describes usingFGFR-Ig fusion proteins in a method of identifying agonists andantagonists of FGFR. However, construction and use of the specificFGFR-Ig fusion proteins as antagonists of FGFR has not been suggested.Other examples of receptor-Ig fusion proteins include those described inU.S. Pat. Nos. 5,726,044; 5,707,632; and 5,750,375.

SUMMARY OF THE INVENTION

[0010] The invention is directed at providing oligomerized forms of FGFRas FGFR antagonists, constructed by fusing extracellular domains of FGFRwith heterologous oligomerization domains. Compositions comprisingpolypeptides and polynucleotides encoding the fusion polypeptides areprovided, as well as methods of using the compositions for treatingdisorders mediated by FGF, FGFR or angiogenesis, such as cancer andother hyperproliferative diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 depicts in vitro binding of ¹²⁵I-bFGF to FGFR. FGFRextracellular domain monomer, FGFR extracellular domain-Ig Fc fusiondimer, and bFGF are compared as competitors of ¹²⁵I-bFGF binding toimmobilized FGFR receptors. Bound ¹²⁵I-bFGF is plotted against a rangeof concentrations of each competitor.

[0012]FIG. 2 depicts binding of ¹²⁵I-bFGF to FGFR: whole cells. FGFRextracellular domain monomer and FGFR extracellular domain-Ig Fc fusiondimer are compared as competitors of ¹²⁵I-bFGF for binding to stableHEK293 cell lines overexpressing FGFR1. Bound ¹²⁵I-bFGF is plottedagainst a range of concentrations of each competitor.

[0013]FIG. 3 depicts bFGF/serum stimulated HUVEC proliferation. FGFRextracellular domain-Ig Fc fusion dimer is tested for its ability toinhibit proliferation of human umbilical vein endothelial cells (HUVEC)in serum- and bFGF-containing media. Optical density (OD) indicatinggrowth is plotted against time after dosing.

[0014]FIG. 4 depicts a comparison of FGFR extracellular domain monomerand FGFR extracellular domain-Ig Fc dimmer on HUVEC proliferation. FGFRextracellular domain monomer and FGFR extracellular domain-Ig Fc fusiondimer are compared for their ability to inhibit HUVEC proliferation atindicated doses.

[0015]FIG. 5 depicts a comparison of FGFR extracellular domain-Ig Fc toanti-bFGF Mab. FGFR extracellular domain-Ig Fc fusion protein and ananti-bFGF monoclonal antibody (Mab) are compared as competitors of¹²⁵I-bFGF binding to immobilized FGF receptors. Bound ¹²⁵I-bFGF isplotted against a range of concentrations of each competitor.

DETAILED DESCRIPTION OF THE INVENTION

[0016] Compositions

[0017] The invention is based on means to provide potent FGF receptor(FGFR) antagonists. Dimeric or higher-order oligomeric forms of FGF arerequired for activation of FGFR, which proceeds through receptordimerization (Moy et al. (1997) Biochemistry 36:4782-4791; Pontaliano etal. (1994) Biochemistry 33:10229-10248). Accordingly, the inventionprovides polypeptides capable of forming oligomers of FGFR, particularlydimers. The polypeptides comprise FGFR extracellular domains fused toheterologous oligomerization domains. Such oligomerized FGF receptorsare provided as FGFR antagonists with higher potency relative to themonomeric FGFR antagonist. More particularly, the invention providespolypeptides comprising specific FGFR extracellular domains fused toregions of immunoglobulin (Ig) molecules which are known to be capableof forming oligomers with other Ig regions.

[0018] “FGFR extracellular domain” as used herein includes that portionof FGFR that is extracellular in native transmembrane forms of thereceptor, or is of such extracellular origin, or consists of all or partof the naturally secreted forms of the receptor. It is understood,however, that the extracellular domain could contain other regions ofthe FGFR receptor (i.e., non-extracellular portions) as long as theseportions do not interfere with or-significantly alter the function ofthe extracellular domain that is relevant to the methods describedherein.

[0019] The FGFR extracellular domain shares homology with theimmunoglobulin supergene family. The FGFR extracellular domain containsIg-like segments. Furthermore, extracellular domains of differentmembers of the FGFR family contain different numbers of Ig-likesegments. The Ig-like segments are classified by their position relativeto the amino-terminus of the FGF receptor, and by sequence homology toknown Ig-like domains. Such classifications are known by a person ofordinary skill in the art. See, for example, Pontaliano et al. (1993)Biochemistry 33:10229-10248; Note 1, 10229.

[0020] The Ig-like segments are generally designated by numberingaccording to the relative positions of the segments from the aminoterminus of FGFR with three Ig-like domains. Such designations are knownby a person of ordinary skill in the art and are used for the purposesof this application, unless indicated otherwise. For example, seePontaliano et al. (1994) Biochemistry 33:10229-10248. Accordingly, informs of FGFR having three Ig-like segments, the Ig I segment isclassified as the first Ig-like segment of an FGFR extracellular domainfrom the amino terminus of the molecule, the Ig II segment as the secondand the Ig III segment as the third.

[0021] In naturally or artificially truncated forms of FGFR which haveless than three Ig-like segments by virtue of the truncation of theextracellular domain, the numbering of the Ig-like segments are retainedas they were prior to the truncation. The numbers designating theIg-like segments are not reassigned according to relative positions ofthe segments from the amino-terminus subsequent to the truncation. Suchdesignations are known in the art and are used for the purposes of thisapplication, unless indicated otherwise. For example, a truncated formof FGFR containing two Ig-like domains due to deletion of a regionencompassing the Ig I segment, is referred to as containing the Ig IIand Ig III segments, although in this truncated form, the Ig II segmentis the first Ig-like segment from the amino-terminus and the Ig IIIsegment is the second.

[0022] Extracellular domains of FGFR containing different numbers andtypes of such Ig-like segments are capable of binding various forms ofFGF. The affinity and specificity of this binding is at least partiallyattributable to the type of Ig-like segment contained within theextracellular domain.

[0023] Thus, the invention relates to providing variants of theextracellular domain of FGFR such that FGFR antagonists can be createdbased on FGF binding capability, affinity, and specificity.

[0024] By “variants” is intended substantially similar sequences. Thus,for nucleotide sequences, variants include those sequences that encodecorresponding parts of the fusion polypeptides of the invention, butthat differ conservatively because of the degeneracy of the geneticcode. These naturally occurring allelic variants can be identified withthe use of well-known molecular biology techniques, such as polymerasechain reaction (PCR) and hybridization techniques as outlined below.

[0025] Variant nucleotide sequences also include synthetically derivednucleotide sequences that have been generated, for example, by usingsite-directed mutagenesis but which still encode corresponding parts ofthe fusion polypeptide sequences provided in the present invention asdiscussed below. Generally, nucleotide sequence variants of theinvention will have at least 70%, preferably at least 80%, morepreferably about 90 to 95% or more, and most preferably about 98% ormore sequence identity to the provided nucleotide sequence.

[0026] A variant polypeptide can differ in amino acid sequence by one ormore substitutions, deletions, insertions, inversions, fusions, andtruncations, or a combination thereof. With respect to the amino acidsequences for various domains of the fusion polypeptides, variantsinclude those domains that are derived from corresponding native domainsby deletion (so-called truncation) or addition of one or more aminoacids to the N-terminal and/or C-terminal end of the polypeptide;deletion or addition of one or more amino acids at one or more sites inthe polypeptide; or substitution of one or more amino acids at one ormore sites in the polypeptide. Such variants may result from, forexample, genetic polymorphism or from human manipulation. Methods forsuch manipulations are generally known in the art.

[0027] Variants of the FGFR extracellular domain include deletionvariants. In one embodiment, amino acid segments that do not contributeto the desired capability, affinity, and specificity of binding of FGFare deleted, while those segments that contribute favorably to thesefunctional properties are retained.

[0028] Deletion variants also include those in which deletion ofparticular amino acid segments positively affects the desired affinityand binding of FGF.

[0029] Deletion could comprise any segment of the extracellular domainincluding but not limited to the Ig I segment. In this aspect, theinvention provides FGFR extracellular domain fusion polypeptides whichlack the Ig I segment. Natural variants of FGFR containing the Ig Isegment and those which lack the Ig I segment are capable of bindingacidic FGF (aFGF) and basic FGF (bFGF) (Kiefer et al. (1991) GrowthFactors 5:115-127; Johnson and Williams (1993) Adv. Cancer. Res.60:1-41). Thus, the Ig I segment is not necessary for binding of aFGFand bFGF. The Ig I deletion further increases the affinity for aFGF andheparin, protects the core of the molecule from proteolysis, andabrogates the heparin requirement for aFGF binding.

[0030] Deletions can range from portions of a segment to deletion of anentire segment. Further, deletions can include one or more deletions inone or more of the Ig-like segments of the FGFR extracellular domain.

[0031] The invention further provides fusion polypeptides in which theFGFR extracellular domain lacks the acid box segment. The acid boxsegment is a known common feature of the FGFR extracellular domain andis characterized by multiple acidic amino acid motifs. For example, seeKiefer et al. (1991) Growth Factors 5:115-127.

[0032] The Ig II segment is typically defined as the second Ig-likesegment from the amino-terminus of an FGFR with three Ig-like segments.The invention encompasses oligomerized FGFR extracellular domainscomprising polypeptides which are variants with respect to the Ig IIsegment. For example, it is recognized that fusion polypeptides lackingboth Ig I and Ig II extracellular segments may have favorable FGFbinding characteristics with respect to affinity and specificity ofbinding and be useful as an FGFR antagonist.

[0033] The Ig III segment is typically defined as the third Ig-likesegment from the amino-terminus of an FGFR with three Ig-like segments.Sequence variants of the C-terminal half of the Ig III segment areassociated with differential FGF binding affinities and specificities.The IIIc variant of the Ig III segment binds aFGF and bFGF with an equalaffinity, higher than that for FGF-7. The IIIb variant binds aFGF andFGF-7 with an equal affinity, higher than that for bFGF. The IIIavariant binds bFGF with a higher affinity than that for aFGF. The IIIcvariant is the most widely expressed natural variant of the Ig IIIsegment, and its deletion decreases the affinity for all ligands of thereceptor. Further descriptions of the Ig IIIa, Ig IIIb, and Ig IIIcsequence variants are provided in Werner et al. (1992) Mol. and CellBiol. 12:82-88, herein incorporated by reference.

[0034] The invention provides fusion polypeptides comprising sequencevariants of the Ig III segment. The selection of the particular Ig IIIvariants is based on the desired FGF binding affinities andspecificities.

[0035] More preferred is a fusion polypeptide in which the FGFRextracellular domain comprises the IIIc sequence variant of the Ig IIIsegment. The invention encompasses fusion polypeptides in which the FGFRextracellular domain comprises the IIIa or the IIIb sequence variants ofthe Ig III segment.

[0036] The invention further relates to providing a fusion polypeptidein which the FGFR extracellular domain comprises combinations of theabove-described variants, including those with deletions of the Ig Isegment, those with deletions of the acid box segment, and thosecomprising sequence variants of the Ig III segment.

[0037] More specifically, the invention provides human FGFR Iextracellular domain fusion polypeptides having the amino acid sequencesset forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, and SEQ ID NO:12. The polypeptides having the sequences set forthin SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12comprise FGFR extracellular domain deletion variants of human FGFR1;lacking one or more segments as described above. Polynucleotidesequences encoding the above polypeptides are also provided and setforth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, and SEQ ID NO:11.

[0038] The FGF receptors of the invention also encompass polypeptidesand polypeptide fragments that comprise FGFR extracellular domains ofother members of the FGFR family, including but not limited to FGFR2,FGFR3, and FGFR4 (Kiefer et al. (1991) Growth Factors 5:115-127).

[0039] The invention also encompasses FGFR fusion polypeptidescomprising other FGFR variants. These variants include substantiallyhomologous FGFR proteins encoded by the same genetic locus, i.e., anallelic variant. The variants also include splicing variants of FGFR.The variants also encompass proteins derived from other genetic loci,but having substantial homology to the provided FGFR. The variants alsoinclude proteins substantially homologous to the provided FGFR butderived from another organism (i.e., non-human), i.e., an ortholog. Thevariants also include proteins substantially homologous that areproduced by chemical synthesis. The variants also include proteinssubstantially homologous that are produced by recombinant methods.

[0040] “Homology” refers to the degree of similarity between x and y.The correspondence between the sequence from one form to another can bedetermined by techniques known in the art. For example, they can bedetermined by a direct comparison of the sequence information of thepolynucleotide. Typically, two sequences, either polynucleotide orpolypeptide, are homologous if the sequences exhibit at least 45%sequence identity; more typically, 50% sequence identity; moretypically, 55% sequence identity; more typically, 60% sequence identity;more typically, 65% sequence identity; even more typically, 70% sequenceidentity. Usually, two sequences are homologous if the sequences exhibitat least 75% sequence identity; more usually, 80% sequence identity;even more usually, 85% sequence identity; even more usually, 90%sequence identity; and even more usually, 95% sequence identity.

[0041] Thus, the invention encompasses polynucleotides having 75%,preferably 80%, more preferably 85%, even more preferably 90%, and mostpreferably 95% or greater sequence identity to the polynucleotidesequences set forth in SEQ ID NOS:1, 3, 5, 7, 9, and 11. The inventionfurther encompasses polypeptides having 75%, preferably 80%, morepreferably 85%, even more preferably 90%, and most preferably 95% orgreater sequence identity to the polypeptide sequences set forth in SEQID NOS:2, 4, 6, 8, 10, and 12).

[0042] Alternatively, homology can be determined by hybridization of thepolynucleotides under conditions which form stable duplexes betweenhomologous regions. Stable duplexes are those, for example, which wouldwithstand digestion with a single-stranded specific nuclease(s), such asS₁. Such duplexes can be analyzed by various methods, such as sizedetermination of digested fragments.

[0043] “Hybridization” refers to the association of two nucleic acidsequences to one another by hydrogen bonding. Typically, one sequencewill be fixed to a solid support and the other will be free in solution.Then, the two sequences will be placed in contact with one another underconditions that favor hydrogen bonding. Factors that affect this bondinginclude: the type and volume of solvent; reaction temperature; time ofhybridization; agitation; agents to block the non-specific attachment ofthe liquid phase sequence to the solid support (Denhardt's reagent orBLOTTO); concentration of the sequences; use of compounds to increasethe rate of association of sequences (dextran sulfate or polyethyleneglycol); and the stringency of the washing conditions followinghybridization. See Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual, 2^(nd) ed., Volume 2, chapter 9, pages 9.47 to 9.57.

[0044] “Stringency” refers to conditions in a hybridization reactionthat favor association of very similar sequences over sequences thatdiffer. For example, the combination of temperature and saltconcentration should be chosen that is approximately 12° C. to 20° C.below the calculated T_(m) of the hybrid under study. The temperatureand salt conditions can often be determined empirically in preliminaryexperiments in which samples of genomic DNA immobilized on filters arehybridized to the sequence of interest and then washed under conditionsof different stringencies. See Sambrook et al., above, at page 9.50.

[0045] Variables to consider when performing, for example, a Southernblot are (1) the complexity of the DNA being blotted and (2) thehomology between the probe and the sequences being detected. The totalamount of the fragment(s) to be studied can vary a magnitude of 10, from0.1 to 1 μg for a plasmid or phage digest to 10⁻⁹ to 10⁻⁸ μg for asingle copy gene in a highly complex eukaryotic genome. For lowercomplexity polynucleotides, substantially shorter blotting,hybridization, and exposure times, a smaller amount of startingpolynucleotides, and lower specific activity of probes can be used. Forexample, a single-copy yeast gene can be detected with an exposure timeof only 1 hour starting with 1 μg of yeast DNA, blotting for two hours,and hybridizing for 4-8 hours with a probe of 10⁸ cpm/μg. For asingle-copy mammalian gene a conservative approach would start with 10μg of DNA, blot overnight, and hybridize overnight in the presence of10% dextran sulfate using a probe of greater than 10⁸ cpm/μg, resultingin an exposure time of ˜24 hours.

[0046] Several factors can affect the melting temperature (T_(m)) of aDNA-DNA hybrid between the probe and the fragment of interest, andconsequently, the appropriate conditions for hybridization and washing.In many cases the probe is not 100% homologous to the fragment. Othercommonly encountered variables include the length and total G+C contentof the hybridizing sequences and the ionic strength and formamidecontent of the hybridization buffer. The effects of all of these factorscan be approximated by a single equation:

T _(m)=81+16.6(log 10C _(i))+0.4[% G+C)]−0.6(% formamide)−600/n−1.5 (%mismatch)

[0047] where C_(i) is the salt concentration (monovalent ions) and n isthe length of the hybrid in base pairs (slightly modified from Meinkothand Wahl (1984) Anal. Biochem. 138:267-284).

[0048] In designing a hybridization experiment, some factors affectingnucleic acid hybridization can be conveniently altered. The temperatureof the hybridization and washes and the salt concentration during thewashes are the simplest to adjust. As the temperature of thehybridization increases (i.e., stringency), it becomes less likely forhybridization to occur between strands that are nonhomologous, and as aresult, background decreases. If the radiolabeled probe is notcompletely homologous with the immobilized fragment (as is frequentlythe case in gene family and interspecies hybridization experiments), thehybridization temperature must be reduced, and background will increase.The temperature of the washes affects the intensity of the hybridizingband and the degree of background in a similar manner. The stringency ofthe washes is also increased with decreasing salt concentrations.

[0049] In general, convenient hybridization temperatures in the presenceof 50% formamide are 42° C. for a probe which is 95% to 100% homologousto the target fragment, 37° C. for 90% to 95% homology, and 32° C. for85% to 90% homology. For lower homologies, formamide content should belowered and temperature adjusted accordingly, using the equation above.If the homology and between the probe and the target fragment are notknown, the simplest approach is to start with both hybridization andwash conditions which are nonstringent. If nonspecific bands or highbackground are observed after autoradiography, the filter can be washedat high stringency and reexposed. If the time required for exposuremakes this approach impractical, several hybridization and/or washingstringencies should be tested in parallel.

[0050] To determine the percent homology of two amino acid sequences, orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of one protein ornucleic acid for optimal alignment with the other protein or nucleicacid). The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in one sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the other sequence, then themolecules are homologous at that position. As used herein, amino acid ornucleic acid “homology” is equivalent to amino acid or nucleic acid“identity.” The percent homology between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent homology equals the number of identical positions/total numberof positions times 100).

[0051] The invention also encompasses fusion polypeptides having a lowerdegree of identity than those described above, but having sufficientsimilarity so as to perform one or more of the same functions performedby the fusion polypeptides of the invention. Similarity is determined byconserved amino acid substitution. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Conservative substitutions are likely to bephenotypically silent. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr;exchange of the acidic residues Asp and Glu; substitution between theamide residues Asn and Gln; exchange of the basic residues Lys and Arg;and replacements among the aromatic residues Phe, Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent are found in Bowie et al. (1990) Science 247:1306-1310. TABLE 1Conservative Amino Acid Substitutions. Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine

[0052] Both identity and similarity can be readily calculated(Computational Molecular Biology, ed. Lesk (Oxford University Press, NewYork, 1988); Biocomputing: Informatics and Genome Projects, ed. Smith(Academic Press, New York, 1993); Computer Analysis of Sequence Data,Part 1, ed. Griffin and Griffin (Humana Press, New Jersey, 1994); vonHeinje (1987) Sequence Analysis in Molecular Biology (Academic Press);and Sequence Analysis Primer, ed. Gribskov and Devereux (M. StocktonPress, New York, 1991).

[0053] Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, GCGprogram package (Devereux et al. (1984) Nucleic Acids Res. 12(1):387),BLASTP, BLASTN, FASTA (Atschul et al. (1990) J. Molec. Biol. 215:403).

[0054] A variant can differ in amino acid sequence by one or moresubstitutions, deletions, insertions, inversions, fusions, andtruncations, or a combination of any of these.

[0055] Variant polypeptides can be fully functional or can lack functionin one or more activities. Thus, in the present case, functions affectedby variations include FGF binding capability, FGF binding affinity, FGFbinding specificity, heparin binding, inhibition of FGF-stimulated cellproliferation, inhibition of FGF-mediated disorders, inhibition ofFGFR-mediated disorders, inhibition of angiogenesis-mediated disorders,and inhibition of cancer and other hyperproliferative disorders.

[0056] Fully functional variants typically contain only conservativevariation or variation in non-critical residues or in non-criticalregions. Functional variants can also contain substitution of similaramino acids which result in no change or an insignificant change infunction. Alternatively, such substitutions may positively or negativelyeffect function to some degree.

[0057] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0058] As indicated, variants can be naturally occurring or can be madeby recombinant means or chemical synthesis to provide useful and novelcharacteristics for the receptor polypeptide. This includes preventingimmunogenicity from pharmaceutical formulations by preventing proteinaggregation.

[0059] Useful variations include alteration of ligand bindingcharacteristics. For example, one specific embodiment encompasses fusionpolypeptide dimers that bind aFGF and bFGF with equal affinity. Afurther embodiment encompasses fusion polypeptide dimers that bind aFGFand bFGF with equal affinity, but with higher than that for FGF-7.Another embodiment encompasses fusion polypeptide dimers that bind aFGFand bFGF with high affinity.

[0060] Another useful variation is one that provides for a proteasecleavage site between the extracellular domain of FGFR and the Igportion of the fusion polypeptide. While constructs containing thecleavage sites are not suited for in vivo use due to the presence of thecleavage site, they are useful as experimental controls. One resultingproduct from utilizing this cleavage site is FGFR extracellular domainmonomer, which is useful as a control in assessing the functionalcharacteristics of the corresponding FGFR extracellular domain-Ig dimerwithout the cleavage site.

[0061] The invention provides polypeptides comprising FGFR extracellulardomains fused to heterologous oligomerization domains. By “heterologousoligomerization domain” is intended that domain of a polypeptide of theinvention which is not naturally associated with the extracellulardomain of the polypeptide, and is capable of forming oligomers, whichare at least dimers, with other polypeptides. Specific examples of suchheterologous oligomerization domains include, but are not limited to,the Fc region of an immunoglobulin molecule; the hinge region of animmunoglobulin molecule; the CH1 region of an immunoglobulin molecule;the CH2 region of an immunoglobulin molecule; the CH3 region of animmunoglobulin molecule; the CH4 region of an immunoglobulin molecule;the light chain of an immunoglobulin molecule; and a peptide comprisinga leucine zipper motif.

[0062] Leucine zipper motifs are known in the art, and are typicallyfound in some of the DNA-binding proteins. Leucine zippers are formedfrom a region of α-helix that contain at least four leucines, eachleucine separated by six amino acids from one another (i.e.,Leu-X₆-Leu-X₆-Leu-X₆-Leu, where X is any common amino acid). See Devlin(1997) Textbook of Biochemistry with Clinical Correlations (4^(th) ed.,John Wiley and Sons, Inc.), pp. 110-111.

[0063] Examples of utilizing various immunoglobulin domains forconstructing oligomeric fusion proteins are known in the art. See, forexample, EP-A 0464 533 and U.S. Pat. No. 5,726,044, which describefusion proteins comprising immunoglobulin Fc regions. See also U.S. Pat.No. 5,750,375, which describes fusion proteins comprising various heavychain domains, as well as light chain kappa.

[0064] The heavy chain Fc region of an immunoglobulin molecule whichcontains the hinge region, but not the CH1 region of the heavy chain, isuseful in therapy and diagnosis; and its incorporation into a proteinresults, for example, in improved pharmacokinetic properties of theprotein. For example, see EP-A 0232 262. In drug discovery applications,human proteins have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists (Bennett et al.(1995) Journal of Molecular Recognition 8:52-58 and Johanson et al.(1995) The Journal of Biological Chemistry 270, 16:9459-9471). Thisinvention encompasses soluble fusion proteins containing a receptorpolypeptide and various portions of the constant regions of heavy orlight chains of immunoglobulins of various subclass (IgG, IgD, IgM, IgA,IgE). Preferred as immunoglobulin is the constant part of the heavychain of human IgG, particularly IgG1, where dimerization between twoheavy chains takes place at the hinge region. It is recognized thatinclusion of the CH2 and CH3 domains of the Fc region as part of thefusion polypeptide increases the in vivo circulation half-life of thepolypeptide comprising the Fc region, and that of the oligomer or dimercomprising the polypeptide.

[0065] Furthermore, the proteins of the invention are generally designedto eliminate or at least minimize the contribution of the Ig Fc fusionprotein to immunogenic responses. To this end, native or mutated Ig Fcportions are preferred which have low or diminished affinity for Fcreceptors, and have diminished capacities for interaction withcomplement; Duncan and Winter (1988) Nature 332:738-740; Xu et al.(1994) J. Biol. Chem. 269:3469-3474. For example, mutations of aminoacids corresponding to Leu 235 and Pro 331 of human IgG1 to Glu and Serrespectively, are provided. More specifically, these mutations areprovided as set forth in SEQ ID NOS:7 and 8 (Construct #4) and SEQ IDNOS:9 and 10 (Construct #5), and described in more detail in Example 6below.

[0066] In order to express any of the fusion proteins of the inventionin a secreted form, a signal peptide is typically contained at theN-terminus of the fusion protein. Generally, the signal peptide that isnative to the FGFR extracellular domain is comprised by the fusionproteins of the invention. Alternatively, signal peptides that areheterologous with respect to the extracellular domain may be used.

[0067] Heparin is known to be required for optimal FGF binding to FGFR.Thus, in constructing the fusion proteins, the heparin binding site isgenerally retained as part of the FGFR extracellular domain.

[0068] Methods for testing the function of the fusion proteins of theinvention include, but are not limited to, the following methods, hereinincorporated by reference: in vitro and in situ growth factor bindingassays (Pontaliano et al. (1994) Biochemistry 33:10229-10248; Kiefer etal. (1991) Growth Factors 5:115-127; U.S. Pat. No. 5,229,501); cellproliferation assays (U.S. Pat. No. 5,229,501; WST cell proliferationassay, Boehringer Mannheim); in vivo and ex vivo assessments ofangiogenesis (Min et al. (1996) Cancer Res. 56:2428-2433; Bickness etal. (1996) Curr. Opin. Oncol. 8:60-65); and assessments of tumor growth(Kim et al. (1993) Nature 362:841-844; Millauer et al. (1993) Nature367:576-579).

[0069] It is understood that other methodologies associated withparticular pathologies, biological conditions, or processes may beemployed when applicable to testing the fusion proteins of theinvention. Examples of such methodologies include molecular biological,immunochemical, histochemical, and morphological assessments relevant tocell proliferation in restenosis (Wilcox (1993) Am. J. Cardiol.72:88E-95E) and ocular diseases (Gariano et al. (1996) Survey Ophthalm.40:481-490). Additional methodologies for assessing vascular density andtumor growth; biochemical assays utilizing markers of angiogenesis; andin vivo methods of assessing therapeutic effects of antiangiogenicagents disclosed by Bickness et al. (1996) Curr. Opin. Oncol. 8:60-65,may also be employed in testing the fusion proteins of the invention,and are herein incorporated by reference.

[0070] Polynucleotide Constructs and Host Cells

[0071] DNA constructs encoding the FGFR fusion polypeptides of theinvention, DNA constructs capable of expressing the FGFR fusionpolypeptides, and host cells containing or capable of expressing suchconstructs are also provided by the invention.

[0072] Routine techniques for the construction of the vectors comprisingthe fusion proteins of the present invention are well-known to those ofordinary skill in the art and can be found in such references asSambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2^(nd)ed., Cold Spring Harbor, N.Y.). A variety of strategies are availablefor ligating fragments of DNA, the choice of which depends on the natureof the termini of the DNA fragments and which choices can be readilymade by those of skill in the art. Generally, and unless otherwisespecified, the 3′ end of the DNA segment encoding the desired FGFRextracellular domains will be ligated in frame to the 5′ end of a DNAsegment encoding the desired oligomerization domain, such that acontiguous fusion protein is produced upon expression of the ligatedDNA. These strategies may encompass PCR techniques in obtaining ormodifying pertinent DNA segments. Also available to one of ordinaryskill in the art is a variety of host cells for containing andexpressing the desired constructs.

[0073] Constructs include native sequences and variants, as well assequences that hybridize under stringent conditions.

[0074] Methods of Using FGFR-IG Fusions

[0075] Working examples of the invention provide methods of inhibitingFGF-stimulated cell proliferation by administering an FGFR antagonistcomprising a fusion of the extracellular domain of FGFR with the Fcregion of an Ig molecule as the heterologous oligomerization domain inan amount effective to inhibit the proliferation. The invention alsoprovides methods of inhibiting angiogenesis by administering to cellscapable of undergoing angiogenesis the fusion protein in an amounteffective to inhibit angiogenesis. The inhibition could be in vitro orin vivo.

[0076] “Amount effective to inhibit” is intended to mean that amount ofthe fusion protein which prevents or induces a measurable inhibition ofFGF-stimulated cell proliferation. Methods of measuring such inhibitionare known to those skilled in the art and include available commercialkits which are based on measuring numbers of viable cells.

[0077] Where the inhibition is in vivo, the amount effective to inhibitcan induce a concentration of the fusion protein in the target organ ortissue needed to inhibit cell proliferation in the cells of the targetorgan or tissue.

[0078] The invention provides methods of treating disorders which areFGF-mediated, FGFR-mediated, or angiogenesis-mediated. The methodsencompass administering therapeutically effective amounts of thepolypeptides of the invention or vectors comprising polynucleotidesencoding the polypeptides of the invention to a mammal. Theadministration can be alone or in conjunction with other agents,including other inhibitors of angiogenesis, tumorigenesis, anticanceragents, and the like.

[0079] Where the treating of a disorder involves administeringpolynucleotides comprising coding regions comprising the polypeptides ofthe invention, the polynucleotides are provided in expression vectorscapable of expressing the polynucleotides in a particular organism,organ, tissue, or cell type; such that the coding region is operablylinked to the promoter of the expression vector.

[0080] The therapeutic polynucleotides and polypeptides of the presentinvention may be utilized in gene delivery vehicles. The gene deliveryvehicle may be of viral or non-viral origin (see generally, Jolly (1994)Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852;Connelly (1995) Human Gene Therapy 1:185-193; and Kaplitt (1994) NatureGenetics 6:148-153). Gene therapy vehicles for delivery of constructsincluding a coding sequence of a therapeutic of the invention can beadministered either locally or systemically. These constructs canutilize viral or non-viral vector approaches. Expression of such codingsequences can be induced using endogenous mammalian or heterologouspromoters. Expression of the coding sequence can be either constitutiveor regulated.

[0081] The present invention can employ recombinant retroviruses whichare constructed to carry or express a selected nucleic acid molecule ofinterest. Retrovirus vectors that can be employed include thosedescribed in EP 0 415 731; WO 90/07936; WO 94/03622; WO 93/25698; WO93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; Vile andHart (1993) Cancer Res. 53:3860-3864; Vile and Hart (1993) Cancer Res.53:962-967; Ram et al. (1993) Cancer Res. 53:83-88; Takamiya et al.(1993) J. Neurosci. Res. 33:493-503; Baba et al. (1993) J. Neurosurg.79:729-735; U.S. Pat. No. 4,777,127; GB Patent No. 2,200,651; and EP 0345 242. Preferred recombinant retroviruses include those described inWO 91/02805.

[0082] Packaging cell lines suitable for use with the above-describedretroviral vector constructs may be readily prepared (see PCTpublications WO 95/30763 and WO 92/05266), and used to create producercell lines (also termed vector cell lines) for the production ofrecombinant vector particles. Within particularly preferred embodimentsof the invention, packaging cell lines are made from human (such asHT1080 cells) or mink parent cell lines, thereby allowing production ofrecombinant retroviruses that can survive inactivation in human serum.

[0083] The present invention also employs alphavirus-based vectors thatcan function as gene delivery vehicles. Such vectors can be constructedfrom a wide variety of alphaviruses, including, for example, Sindbisvirus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), RossRiver virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equineencephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCCVR-532). Representative examples of such vector systems include thosedescribed in U.S. Pat. Nos. 5,091,309; 5,217,879; and 5,185,440; and PCTNos. WO 92/10578; WO 94/21792; WO 95/27069; WO 95/27044; and WO95/07994.

[0084] Gene delivery vehicles of the present invention can also employparvovirus such as adeno-associated virus (AAV) vectors. Representativeexamples include the AAV vectors disclosed by Srivastava in WO 93/09239,Samulski et al. (1989) J. Vir. 63:3822-3828; Mendelson et al. (1988)Virol. 166:154-165; and Flotte et al. (1993) PNAS 90:10613-10617.

[0085] Representative examples of adenoviral vectors include thosedescribed by Berkner (1988) Biotechniques 6:616-627; Rosenfeld et al.(1991) Science 252:431-434; WO 93/19191; Kolls et al. (1994) PNAS91:215-219; Kass-Eisler et al. (1993) PNAS 90:11498-11502; Guzman et al.(1993) Circulation 88:2838-2848; Guzman et al. (1993) Cir. Res.73:1202-1207; Zabner et al. (1993) Cell 75:207-216; Li et al. (1993)Hum. Gene Ther. 4:403-409; Cailaud et al. (1993) Eur. J. Neurosci.5:1287-1291; Vincent et al. (1993) Nat. Genet. 5:130-134; Jaffe et al.(1992) Nat. Genet. 1:372-378; and Levrero et al. (1991) Gene101:195-202. Exemplary adenoviral gene therapy vectors employable inthis invention also include those described in WO 94/12649; WO 93/03769;WO 93/19191; WO 94/28938; WO 95/11984; and WO 95/00655. Administrationof DNA linked to killed adenovirus as described in Curiel (1992) HumanGene Therapy 3:147-154 maybe employed.

[0086] Other gene delivery vehicles and methods may be employed,including polycationic condensed DNA linked or unlinked to killedadenovirus alone, for example Curiel (1992) Human Gene Therapy3:147-154; ligand linked DNA, for example see Wu (1989) Biol. Chem.264:16985-16987; eukaryotic cell delivery vehicles cells, for examplesee U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No.08/404,796; deposition of photopolymerized hydrogel materials; hand-heldgene transfer particle gun, as described in U.S. Pat. No. 5,149,655;ionizing radiation as described in U.S. Pat. No. 5,206,152 and in WO92/11033; nucleic charge neutralization or fusion with cell membranes.Additional approaches are described in Philip (1994) Mol. Cell Biol.14:2411-2418, and in Woffendin (1994) Proc. Natl. Acad. Sci. USA91:1581-1585.

[0087] Naked DNA may also be employed. Exemplary naked DNA introductionmethods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptakeefficiency may be improved using biodegradable latex beads. DNA coatedlatex beads are efficiently transported into cells after endocytosisinitiation by the beads. The method may be improved further by treatmentof the beads to increase hydrophobicity and thereby facilitatedisruption of the endosome and release of the DNA into the cytoplasm.Liposomes that can act as gene delivery vehicles are described in U.S.Pat. No. 5,422,120; PCT Nos. WO 95/13796, WO 94/23697, and WO 91/14445;and EP No. 0 524 968.

[0088] Further non-viral delivery suitable for use includes mechanicaldelivery systems such as the approach described in Woffendin et al.(1994) Proc. Natl. Acad. Sci. USA 91(24):11581-11585. Moreover, thecoding sequence and the product of expression of such can be deliveredthrough deposition of photopolymerized hydrogel materials. Otherconventional methods for gene delivery that can be used for delivery ofthe coding sequence include, for example, use of hand-held gene transferparticle gun, as described in U.S. Pat. No. 5,149,655; use of ionizingradiation for activating transferred gene, as described in U.S. Pat. No.5,206,152 and PCT No. WO 92/11033.

[0089] The invention also provides a method of diagnosing disordersinvolving FGF-stimulated cell proliferation, angiogenesis andtumorigenesis.

[0090] In one embodiment, the method encompasses administering to testcells an effective amount of the fusion proteins of the invention andassessing inhibition compared to a control.

[0091] In another embodiment of the invention, the above-describedmethods of diagnosis encompass in vitro administration of any of thedescribed fusion protein antagonists to cells in culture.

[0092] In another embodiment of the invention, the above-describedmethods of diagnosis involve administering any of the described fusionproteins in vivo.

[0093] In one embodiment, therapeutically effective amounts ofpharmacological compositions containing fusion protein antagonists asdescribed herein are administered to a patient or an animal model inneed of such administration. The methods of treatment or preventionencompass administering effective amounts of a pharmacologicalcomposition containing a fusion protein of the invention as describedherein.

[0094] Methods of constructing therapeutically effective fusion proteinantagonists are provided. In one embodiment of the invention, bindingaffinities and specificities of the fusion proteins are firstcharacterized in vitro. Next, proteins with desired affinities andspecificities against various forms of FGF are selected and furtherassessed for inhibition of FGF-stimulated cell proliferation. Next, newfusion proteins are constructed by deletion of segments determined notto be necessary for desired affinities and specificities of binding toFGF or high potency of inhibition of FGF-stimulated cell proliferation.The above-described assessments and selections are repeated with thesmaller deletion constructs; until a minimal protein structure havingthe desired affinities, specificities and potencies is constructed. Thetherapeutic effectiveness of selected minimal constructs are thenassessed in vivo.

[0095] The administration includes, but is not limited to,administration to animal models and patients manifesting the followingdisorders: restenosis after angioplasty or atherectomy (Wilcox (1993)Am. J. Cardiol. 72:88E-95E), ophthalmological disorders involvingexcessive vasoproliferation (Gariano et al. (1996) Survey Ophthalm.40:481-490), various tumors and cancers (Kim et al. (1993) Nature334:841-844; Kim et al. (1993) Nature 362:841-844; Min et al. (1996)Cancer Res. 56:2428-2433) including AIDS-related Kaposi sarcoma(Pontaliano et al. (1994) Biochemistry 33:10229-10248).

[0096] Pharmacological Compositions

[0097] The invention provides pharmacological compositions comprisingthe fusion polypeptides of the invention or polynucleotides encoding thepolypeptides described herein. The pharmacological compositions may alsocontain any of the described variants of FGFR.

[0098] Compounds useful for formulating polypeptides and/or proteinpharmaceutical compositions can be used with fusion proteins.

[0099] The pharmaceutical compositions will comprise a therapeuticallyeffective amount of any of the proteins of the claimed invention.

[0100] The term “therapeutically effective amount” as used herein refersto an amount of a therapeutic agent to treat or prevent a disordersufficient to exhibit a detectable preventive, ameliorative, curative orother therapeutic effect. The effect may include, for example,treatment, amelioration, or prevention of any physical or biochemicalcondition, for example, including but not limited to hyperproliferativegrowth, angiogenesis and cancer.

[0101] The effect can be detected by, for example, biochemical orhistological means of assessing angiogenesis. Therapeutic effects alsoinclude reduction in physical symptoms, such as decreased tumor size.The precise effective amount for a subject will depend upon thesubject's size and health, the nature and extent of the condition, andthe therapeutics or combination of therapeutics selected foradministration. Thus, it is not useful to specify an exact effectiveamount in advance. However, the effective amount for a given situationcan be determined by routine experimentation.

[0102] In this aspect of the invention, by “FGF-mediated disorder,”“FGFR-mediated disorder,” or “angiogenesis-mediated disorder” isintended an adverse biological or biochemical condition that isexacerbated by FGF, FGFR, or angiogenesis. Examples of such disordersinclude, but are not limited to, tumorigenesis, neovascularization,hyper-proliferation of vascular smooth muscle cells, and the like.

[0103] Tumors include, but are not limited to, bladder, breast,node-negative breast, lung, rectal, colorectal, testis, and cervicaltumors; glioblastoma; childhood brain tumors; squamous cancer of thetongue; etc.

[0104] Disorders involving neovascularization include, but are notlimited to, diabetic retinopathy, retinopathy of prematurity (ROP),choroidal neovascularization, neovascular glaucoma, wound healing aftersurgery and injury, corneal scarring, ocular neoplasia, and breakdown ofblood-retina barrier.

[0105] Disorders involving hyperproliferation of vascular smooth musclecells include, but are not limited to, postangioplasty andpostatherectomy restenosis.

[0106] It is recognized that depending on the type and stage of aparticular disorder, the disorder may be mediated by FGF, FGFR,angiogenesis, or combinations thereof.

[0107] For purposes of the present invention, an effective dose will befrom about 1 pg/kg to 10 mg/kg of the fusion protein in the individualto which it is administered.

[0108] A pharmaceutical composition can also contain a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”refers to a carrier for administration of a therapeutic agent, such asantibodies or a polypeptide, genes, and other therapeutic agents. Theterm refers to any pharmaceutical carrier that does not itself inducethe production of antibodies harmful to the individual receiving thecomposition, and which may be administered without undue toxicity.Suitable carriers may be large, slowly metabolized macromolecules suchas proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid coknown to those of ordinary skill inthe art.

[0109] Pharmaceutically acceptable salts can be used therein, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. A thoroughdiscussion of pharmaceutically acceptable excipients are available inRemington 's Pharmaceutical Sciences (Mack Pub. Co., NJ, 1991).

[0110] Pharmaceutically acceptable carriers in therapeutic compositionsmay contain liquids such as water, saline, glycerol and ethanol.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present in suchvehicles. Typically, the therapeutic compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection may also be prepared. Liposomes are included within thedefinition of a pharmaceutically acceptable carrier.

[0111] Administration Methods

[0112] Once formulated, the fusion protein compositions of the inventioncan be (1) administered directly to the subject; or delivered ex vivo,to cells derived from the subject.

[0113] Direct delivery of the compositions will generally beaccomplished by injection, either subcutaneously, intraperitoneally,intravenously, or intramuscularly, or delivered to the interstitialspace of a tissue. The compositions can also be administered into atumor or lesion. Other modes of administration include oral andpulmonary administration, suppositories, and transdermal applications,needles, and gene guns, or hyposprays. Dosage treatment may be a singledose schedule or a multiple dose schedule.

[0114] Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or absorb the compositions. Thecontrolled delivery may be exercised by selecting appropriatemacromolecules (for example, polyesters, polyamino acids, polyvinylpyrrolidone, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate). The rate of drug releasemay also be controlled by altering the concentration of suchmacromolecules.

[0115] Another possible method for controlling the duration of actioncomprises incorporating the therapeutic agents into particles of apolymeric substance such as polyesters, polyamino acids, hydrogels,poly(lactic acid), or ethylene vinylacetate copolymers. Alternatively,it is possible to entrap the therapeutic agents in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, by the use of hydroxymethyl cellulose orgelatin-microcapsules or poly(methylmethacrylate) microcapsules,respectively, or in a colloid drug delivery system, for example,liposomes, albumin, microspheres, microemulsions, nanoparticles,nanocapsules, or in macroemulsions. Such teachings are disclosed inRemington's Pharmaceutical Sciences (1980).

[0116] A preferred controlled release composition for delivery of suchcompositions will encompass liposome encapsulation as described in, butnot limited to U.S. Pat. No. 4,522,803; EP 0 280 503 B1; and WO95/13796.

[0117] Detecting Cell Proliferation

[0118] The invention provides methods of diagnosing FGF-stimulated cellproliferation and angiogenesis by administering to test cells aneffective amount of any of the fusion proteins and assessing inhibitionagainst a control. The cells can be in vivo or in vitro. In this aspect,the administration could be directed at animal models or patients,including those described above, or directed at primary cultures or celllines derived from tissues affected by disease states potentiallyinvolving FGF-stimulation. The method of diagnosis is, thus, useful fordetermining the propensity of an animal to respond to treatment withfusion protein antagonists as provided by the invention.

[0119] In this manner, the invention relates to providing receptoroligomer fusion constructs which can effect clinically determinableinhibition of angiogenesis or diseases involving angiogenesis whenadministered in effective amounts to patients or animal models in needof such administration.

EXPERIMENTAL

[0120] FGFR-Ig Fc fusion protein dimer (FGFR ECD Ig), according toConstruct #1 (Example 6) was characterized with respect to bFGF bindingin vitro (FIG. 1) and in whole cells (FIG. 2). Determined IC₅₀ valuesindicate that the dimerized receptor is an effective antagonist of FGFbinding at subnanomolar concentrations, and is 20-50 fold more potentthan the monomeric form (FGFR ECD mono) in competing for bFGF binding.

Example 1 In Vitro Binding of ¹²⁵I-bFGF to FGFR (FIG. 1)

[0121] FGFR monomer (U.S. Pat. No. 5,229,501) and FGFR-Ig Fc fusiondimer according to Construct #1 (Example 6) were tested for theirability to compete with ¹²⁵I-bFGF for binding to immobilized FGFreceptors. Immulon 2 microtiter plates were coated with 10 nM FGFR-IgFcfusion protein (Construct #1) in 50 mM Na₂CO₃, pH 9.6 overnight at 4° C.The plates were then blocked with 1% gelatin in Dulbecco's PBS (DPBS)for 1 hr at room temperature, and washed with DPBS plus 0.05% Tween 20.Serial dilutions in duplicate of FGFR-Ig fusion dimer (Construct #1,Example 6), FGFR monomer, or bFGF were added, followed by asubsaturating amount (0.5 nM final) ¹²⁵I-bFGF in DPBS plus 0.1% gelatin,0.1% triton X-100, and 10 μM Heparin, pH 7.75. After 2 hr at roomtemperature, wells were washed and the bound radioactivity determined bygamma counting. Displacement curves were analyzed with a four parameterfit to obtain IC₅₀ values. The results indicate that the FGFR-Ig Fcfusion dimer is active as a FGF antagonist at subnanomolarconcentrations, and is approximately 20-fold more potent than the FGFRmonomer protein as a competitor of bFGF binding to immobilized FGFreceptors.

Example 2 Binding of ¹²⁵I-bFGF to FGFR: Whole Cells (FIG. 2)

[0122] FGFR monomer (U.S. Pat. No. 5,229,501) and FGFR-Ig fusion dimeraccording to Construct #1 (Example 6) were tested for their ability tocompete with ¹²⁵I-bFGF for binding to stable HEK293 cell linesoverexpressing FGFR1. FGFR was overexpressed (0.3×10⁶ receptors/cell) inHEK293 cells by transfection of the FGFR1 cDNA (U.S. Pat. No. 5,229,501)in the high copy number plasmid pcDNA3 and selecting clones resistant toG418. 1.5×10⁵ cells were plated in 24 well plates in DMEM plus 10%serum. Following overnight incubation, the cells were washed twice with1 ml DMEM plus 0.2% gelatin and 15 units/ml heparin. Serial dilutions ofFGFR extracellular domain-Fg Fc fusion dimer, FGFR monomer, or bFGF werepre-mixed with 0.1 nM ¹²⁵I-bFGF (1138 Ci/mmol) and 250 μl of thismixture was added to each well, and incubated at 37° C. for 30 minutes.The media was removed, and the cells washed three times with 1 ml DMEMcontaining 150 mM NaCl, 0.2% gelatin, and 15 units/ml heparin. The cellswere lysed in 250 μl 0.1% SDS and lysates counted in a gamma counter.Displacement curves were analyzed with a four parameter fit to obtainIC₅₀ values. In this assay, the FGFR-Ig fusion dimer is approximately50-fold more potent than the FGFR monomer protein as a competitor ofbFGF binding to cells.

Example 3 bFGF/Serum Stimulated HUVEC Proliferation (FIG. 3)

[0123] FGFR-Ig fusion dimer (FGFR ECD Ig), according to Construct #1(Example 6) was tested for its ability to inhibit proliferation of humanumbilical vein endothelial cells (HUVEC) in serum- and bFGF-containingmedia. HUVEC cells were plated in gelatin coated 96-well plates at adensity of 2000 cells/well in 50 μl of EBM (endothelial basalmedia-Clonetics). The cells were incubated overnight (37° C. 5% CO₂),the media was removed and 200 μl of media was added containing 10 ng/mlbFGF+90 μg/ml heparin+either 2% or 5% FBS and +/−0.1 μM FGFR fusion(Construct #1). The FGFR fusion was preincubated with bFGF and heparinfor at least 30 minutes prior to addition to cell wells. Proliferationindexes were determined on days 0, 1, 2, 3, 4 and 5 using the WST-1 cellproliferation assay (Boehringer Mannheim), which measures number ofviable cells. 100 nM FGFR-Ig fusion dimer completely inhibitsbFGF-stimulated HUVEC proliferation, even in the presence of 5% serum.

Example 4 HUVEC (Proliferation: Comparison of FGFR Monomer and FGFR-IgDimer FIG. 4)

[0124] FGFR monomer (U.S. Pat. No. 5,229,501) and FGFR-Ig fusion dimer(FGFR ECD Ig), according to Construct #1 (Example 6) were compared fortheir ability to inhibit HUVEC proliferation at different doses in thepresence of 10 ng/ml bFGF+90 μg/ml heparin+5% FBS as described inExample 3 above. The FGFR-Ig fusion dimer is more than 10-fold morepotent than the FGFR monomer as an inhibitor of HUVEC proliferation inthe presence of serum and bFGF, can inhibit proliferation completely,and can inhibit proliferation even below the level seen in the absenceof FGF. These results are consistent with the data from in vitro andwhole-cell binding assays of FIGS. 1-3.

[0125]FIGS. 3 and 4 indicate that not only FGFR-Ig is a more potentinhibitor of this proliferation than monomeric extracellular domains ofFGFR, the fusion protein is capable of 100% inhibition of theproliferation.

Example 5 Comparison of FGFR-Ig to Anti-bFGF Mab (FIG. 5)

[0126] The ability of the FGFR-Ig fusion dimer (FGFR ECD Ig), accordingto Construct #1 (Example 6) to compete with ¹²⁵I-bFGF for binding toimmobilized receptors was compared to the highest affinity neutralizingmouse monoclonal antibody to bFGF which is available (UpstateBiotechnology Inc). The assay was performed as in (A). The overall IC₅₀sfor these two protein is similar, but the antibody displays somenon-competitive binding behavior, and is unable to completely inhibit¹²⁵I-bFGF binding, even at high concentrations. In contrast, the FGFR-Igfusion protein completely inhibits binding.

Example 6 FGFR Extracellular Domain-Ig Fc Fusion Constructs

[0127] SEQ ID NOS:1-12 set forth nucleotide and amino acid sequences forfusion protein Constructs #1-6 comprising segments of an FGFRextracellular domain fused to the Fc region of an immunoglobulinmolecule.

[0128] Construct #1

[0129] The polynucleotide and amino acid sequences of Construct #1 areset forth in SEQ ID NOS:1 and 2, respectively.

[0130] Construct #1 contains, in order from the 5′/NH2-terminus to the3′/COOH-terminus: human FGFR1 signal peptide (comprised by amino acids1-21); human FGFR1 extracellular domain (nucleotides 64-1123, aminoacids 22-374) which contains the Ig I segment (comprised by nucleotides163-303, amino acids 55-101), the acid box segment (nucleotides 376-399,amino acids 126-133), the Ig II segment (nucleotides 526-684, aminoacids 176-228), and the IIIc variant of Ig III segment (nucleotides823-1017, amino acids 275-339); a linker sequence with a thrombincleavage site (nucleotides 1123-1170, amino acids 375-390); and the Fcportion of human IgG1 which includes the hinge region, and domains CH2and CH3 (nucleotides 1171-1869, amino acids 391-622).

[0131] Construct #2

[0132] The polynucleotide and amino acid sequences of Construct #2 areset forth in SEQ ID NOS:3 and 4, respectively.

[0133] Construct #2 comprises deleting from Construct #1 the Ig Isegment plus additional flanking sequences (nucleotides 91-357, aminoacids 31-119, as numbered in Construct #1), and part of the linkerencompassing the thrombin cleavage site (nucleotides 1123-1146, aminoacids 375-382, as numbered in Construct #1).

[0134] Accordingly, Construct #2 comprises from the 5′/NH2-terminus tothe 3′/COOH-terminus: human FGFR1 signal peptide, human FGFR1extracellular domain which contains the acid box segment, the Ig IIsegment and the IIIc variant of Ig III segment; and the Fc portion ofhuman IgG1 which includes the hinge region, and domains CH2 and CH3.

[0135] Construct #3

[0136] The polynucleotide and amino acid sequences of Construct #3 areset forth in SEQ ID NOS:5 and 6, respectively.

[0137] Construct #3 comprises deleting from Construct #1 the Ig Isegment as well as the acid box and flanking sequences (nucleotides91-441, amino acids 31-147, as numbered in Construct #1), and part ofthe linker encompassing the thrombin cleavage site (nucleotides1123-1146, amino acids 375-382, as numbered in Construct #1).

[0138] Accordingly, Construct #3 comprises from the 5′/NH2-terminus tothe 3′/COOH-terminus: human FGFR1 signal peptide, human FGFR1extracellular domain which contains the Ig II segment and the IIIcvariant of the Ig III segment; and the Fc portion of human IgG1 whichincludes the hinge region, and domains CH2 and CH3.

[0139] Construct #4

[0140] The polynucleotide and amino acid sequences of Construct #4 areset forth in SEQ ID NOS:7 and 8, respectively.

[0141] Construct #4 is the same as Construct #2 with two additionalchanges:

[0142] a) Nucleotides 937 to 938 were changed from “CT” to “GA”, whichchanges amino acid 313 from LEU to GLU (as numbered in Constructs #2 and4). This mutation decreases the affinity of the Fc portion for Fcreceptors.

[0143] b) Nucleotide 1225 was changed from “C” to “T”, which changesamino acid 409 from PRO to SER (as numbered in sequences #2 and 4). Thismutation decreases the affinity of the Fc portion for complement.

[0144] The positions correspond to amino acids 235 (LEU to GLU) and 331(Pro to Ser) of human IgG1.

[0145] Construct #5

[0146] The polynucleotide and amino acid sequences of Construct #5 areset forth in SEQ ID NOS:9 and 10, respectively.

[0147] Construct #5 is the same as Construct #3 with two additionalchanges:

[0148] a) Nucleotides 853 to 854 have been changed from “CT” to “GA”,which changes amino acid 285 from LEU to GLU (as numbered in sequences#3 and 5).

[0149] b) Nucleotide 1141 has been changed from “C” to “T”, whichchanges amino acid 385 from PRO to SER.

[0150] The positions changed correspond to amino acids 235 (LEU to GLU)and 331 (Pro to Ser) of human IgG1, and result in decreased affinitiesof the Fc portion for Fc receptors and complement respectively.

[0151] Construct #6

[0152] The polynucleotide and amino acid sequences of Construct #6 areset forth in SEQ ID NOS:11 and 12, respectively.

[0153] Construct #6 is the same as Construct #5 with one change:Nucleotides 772-798 (amino acids 258-266), as numbered in Construct #5,were deleted. Thus, this construct lacks all the residues from thelinker sequence encompassing the thrombin cleavage site as described inConstruct #1, and is potentially the least immunogenic construct.

[0154] The purpose of this construct was to eliminate all residual aminoacids left over from the original linker segment in Construct #1.Construct #6 has the same activity as the other constructs and is thepreferred construct for administration as a therapeutic, because it ispotentially the least immunogenic construct.

[0155] Other modifications and embodiments of the invention will come tomind in one skilled in the art to which this invention pertains havingthe benefit of the teachings presented herein. Therefore, it is to beunderstood that the invention is not to be limited to the specificembodiments disclosed. Although specific terms are employed, they areused in generic and descriptive sense only and not for purposes oflimitation, and that modifications and embodiments are intended to beincluded within the scope of the appended claims.

1 12 1 1869 DNA Homo sapiens CDS (1)...(1869) 1 atg tgg agc tgg aag tgcctc ctc ttc tgg gct gtg ctg gtc aca gcc 48 Met Trp Ser Trp Lys Cys LeuLeu Phe Trp Ala Val Leu Val Thr Ala 1 5 10 15 aca ctc tgc acc gct aggccg tcc ccg acc ttg cct gaa caa gcc cag 96 Thr Leu Cys Thr Ala Arg ProSer Pro Thr Leu Pro Glu Gln Ala Gln 20 25 30 ccc tgg gga gcc cct gtg gaagtg gag tcc ttc ctg gtc cac ccc ggt 144 Pro Trp Gly Ala Pro Val Glu ValGlu Ser Phe Leu Val His Pro Gly 35 40 45 gac ctg ctg cag ctt cgc tgt cggctg cgg gac gat gtg cag agc atc 192 Asp Leu Leu Gln Leu Arg Cys Arg LeuArg Asp Asp Val Gln Ser Ile 50 55 60 aac tgg ctg cgg gac ggg gtg cag ctggcg gaa agc aac cgc acc cgc 240 Asn Trp Leu Arg Asp Gly Val Gln Leu AlaGlu Ser Asn Arg Thr Arg 65 70 75 80 atc aca ggg gag gag gtg gag gtg caggac tcc gtg ccc gca gac tcc 288 Ile Thr Gly Glu Glu Val Glu Val Gln AspSer Val Pro Ala Asp Ser 85 90 95 ggc ctc tat gct tgc gta acc agc agc ccctcc gga agt gac acc acc 336 Gly Leu Tyr Ala Cys Val Thr Ser Ser Pro SerGly Ser Asp Thr Thr 100 105 110 tac ttc tcc gtc aat gtt tca gat gct ctcccc tcc tcg gag gat gat 384 Tyr Phe Ser Val Asn Val Ser Asp Ala Leu ProSer Ser Glu Asp Asp 115 120 125 gat gat gat gat gac tcc tct tca gag gagaaa gaa aca gat aac acc 432 Asp Asp Asp Asp Asp Ser Ser Ser Glu Glu LysGlu Thr Asp Asn Thr 130 135 140 aaa cca aac ccc gta gct cca tat tgg acatcc cca gaa aag atg gaa 480 Lys Pro Asn Pro Val Ala Pro Tyr Trp Thr SerPro Glu Lys Met Glu 145 150 155 160 aag aaa ttg cat gca gtg ccg gct gccaag aca gtg aag ttc aaa tgc 528 Lys Lys Leu His Ala Val Pro Ala Ala LysThr Val Lys Phe Lys Cys 165 170 175 cct tcc agt ggg acc cca aac ccc acactg cgc tgg ttg aaa aat ggc 576 Pro Ser Ser Gly Thr Pro Asn Pro Thr LeuArg Trp Leu Lys Asn Gly 180 185 190 aaa gaa ttc aaa cct gac cac aga attgga ggc tac aag gtc cgt tat 624 Lys Glu Phe Lys Pro Asp His Arg Ile GlyGly Tyr Lys Val Arg Tyr 195 200 205 gcc acc tgg agc atc ata atg gac tctgtg gtg ccc tct gac aag ggc 672 Ala Thr Trp Ser Ile Ile Met Asp Ser ValVal Pro Ser Asp Lys Gly 210 215 220 aac tac acc tgc att gtg gag aat gagtac ggc agc atc aac cac aca 720 Asn Tyr Thr Cys Ile Val Glu Asn Glu TyrGly Ser Ile Asn His Thr 225 230 235 240 tac cag ctg gat gtc gtg gag cggtcc cct cac cgg ccc atc ctg caa 768 Tyr Gln Leu Asp Val Val Glu Arg SerPro His Arg Pro Ile Leu Gln 245 250 255 gca ggg ttg ccc gcc aac aaa acagtg gcc ctg ggt agc aac gtg gag 816 Ala Gly Leu Pro Ala Asn Lys Thr ValAla Leu Gly Ser Asn Val Glu 260 265 270 ttc atg tgt aag gtg tac agt gacccg cag ccg cac atc cag tgg cta 864 Phe Met Cys Lys Val Tyr Ser Asp ProGln Pro His Ile Gln Trp Leu 275 280 285 aag cac atc gag gtg aat ggg agcaag att ggc cca gac aac ctg cct 912 Lys His Ile Glu Val Asn Gly Ser LysIle Gly Pro Asp Asn Leu Pro 290 295 300 tat gtc cag atc ttg aag act gctgga gtt aat acc acc gac aaa gag 960 Tyr Val Gln Ile Leu Lys Thr Ala GlyVal Asn Thr Thr Asp Lys Glu 305 310 315 320 atg gag gtg ctt cac tta agaaat gtc tcc ttt gag gac gca ggg gag 1008 Met Glu Val Leu His Leu Arg AsnVal Ser Phe Glu Asp Ala Gly Glu 325 330 335 tat acg tgc ttg gcg ggt aactct atc gga ctc tcc cat cac tct gca 1056 Tyr Thr Cys Leu Ala Gly Asn SerIle Gly Leu Ser His His Ser Ala 340 345 350 tgg ttg acc gtt ctg gaa gccctg gaa gag agg ccg gca gtg atg acc 1104 Trp Leu Thr Val Leu Glu Ala LeuGlu Glu Arg Pro Ala Val Met Thr 355 360 365 tcg ccc ctg tac ctg gag tctaga ggt ggt cta gtg ccg cgc ggc agc 1152 Ser Pro Leu Tyr Leu Glu Ser ArgGly Gly Leu Val Pro Arg Gly Ser 370 375 380 ggt tcc ccc ggg ttg cag gagccc aaa tct tgt gac aaa act cac aca 1200 Gly Ser Pro Gly Leu Gln Glu ProLys Ser Cys Asp Lys Thr His Thr 385 390 395 400 tgc cca ccg tgc cca gcacct gaa ctc ctg ggg gga ccg tca gtc ttc 1248 Cys Pro Pro Cys Pro Ala ProGlu Leu Leu Gly Gly Pro Ser Val Phe 405 410 415 ctc ttc ccc cca aaa cccaag gac acc ctc atg atc tcc cgg acc cct 1296 Leu Phe Pro Pro Lys Pro LysAsp Thr Leu Met Ile Ser Arg Thr Pro 420 425 430 gag gtc aca tgc gtg gtggtg gac gtg agc cac gaa gac cct gag gtc 1344 Glu Val Thr Cys Val Val ValAsp Val Ser His Glu Asp Pro Glu Val 435 440 445 aag ttc aac tgg tac gtggac ggc gtg gag gtg cat aat gcc aag aca 1392 Lys Phe Asn Trp Tyr Val AspGly Val Glu Val His Asn Ala Lys Thr 450 455 460 aag ccg cgg gag gag cagtac aac agc acg tac cgt gtg gtc agc gtc 1440 Lys Pro Arg Glu Glu Gln TyrAsn Ser Thr Tyr Arg Val Val Ser Val 465 470 475 480 ctc acc gtc ctg caccag gac tgg ctg aat ggc aag gag tac aag tgc 1488 Leu Thr Val Leu His GlnAsp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 485 490 495 aag gtc tcc aac aaagcc ctc cca gcc ccc atc gag aaa acc atc tcc 1536 Lys Val Ser Asn Lys AlaLeu Pro Ala Pro Ile Glu Lys Thr Ile Ser 500 505 510 aaa gcc aaa ggg cagccc cga gaa cca cag gtg tac acc ctg ccc cca 1584 Lys Ala Lys Gly Gln ProArg Glu Pro Gln Val Tyr Thr Leu Pro Pro 515 520 525 tcc cgg gat gag ctgacc aag aac cag gtc agc ctg acc tgc ctg gtc 1632 Ser Arg Asp Glu Leu ThrLys Asn Gln Val Ser Leu Thr Cys Leu Val 530 535 540 aaa ggc ttc tat cccagc gac atc gcc gtg gag tgg gag agc aat ggg 1680 Lys Gly Phe Tyr Pro SerAsp Ile Ala Val Glu Trp Glu Ser Asn Gly 545 550 555 560 cag ccg gag aacaac tac aag acc acg cct ccc gtg ctg gac tcc gac 1728 Gln Pro Glu Asn AsnTyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 565 570 575 ggc tcc ttc ttcctc tac agc aag ctc acc gtg gac aag agc agg tgg 1776 Gly Ser Phe Phe LeuTyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 580 585 590 cag cag ggg aacgtc ttc tca tgc tcc gtg atg cat gag gct ctg cac 1824 Gln Gln Gly Asn ValPhe Ser Cys Ser Val Met His Glu Ala Leu His 595 600 605 aac cac tac acgcag aag agc ctc tcc ctg tct ccg ggt aaa tga 1869 Asn His Tyr Thr Gln LysSer Leu Ser Leu Ser Pro Gly Lys * 610 615 620 2 622 PRT Homo sapiens 2Met Trp Ser Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val Thr Ala 1 5 1015 Thr Leu Cys Thr Ala Arg Pro Ser Pro Thr Leu Pro Glu Gln Ala Gln 20 2530 Pro Trp Gly Ala Pro Val Glu Val Glu Ser Phe Leu Val His Pro Gly 35 4045 Asp Leu Leu Gln Leu Arg Cys Arg Leu Arg Asp Asp Val Gln Ser Ile 50 5560 Asn Trp Leu Arg Asp Gly Val Gln Leu Ala Glu Ser Asn Arg Thr Arg 65 7075 80 Ile Thr Gly Glu Glu Val Glu Val Gln Asp Ser Val Pro Ala Asp Ser 8590 95 Gly Leu Tyr Ala Cys Val Thr Ser Ser Pro Ser Gly Ser Asp Thr Thr100 105 110 Tyr Phe Ser Val Asn Val Ser Asp Ala Leu Pro Ser Ser Glu AspAsp 115 120 125 Asp Asp Asp Asp Asp Ser Ser Ser Glu Glu Lys Glu Thr AspAsn Thr 130 135 140 Lys Pro Asn Pro Val Ala Pro Tyr Trp Thr Ser Pro GluLys Met Glu 145 150 155 160 Lys Lys Leu His Ala Val Pro Ala Ala Lys ThrVal Lys Phe Lys Cys 165 170 175 Pro Ser Ser Gly Thr Pro Asn Pro Thr LeuArg Trp Leu Lys Asn Gly 180 185 190 Lys Glu Phe Lys Pro Asp His Arg IleGly Gly Tyr Lys Val Arg Tyr 195 200 205 Ala Thr Trp Ser Ile Ile Met AspSer Val Val Pro Ser Asp Lys Gly 210 215 220 Asn Tyr Thr Cys Ile Val GluAsn Glu Tyr Gly Ser Ile Asn His Thr 225 230 235 240 Tyr Gln Leu Asp ValVal Glu Arg Ser Pro His Arg Pro Ile Leu Gln 245 250 255 Ala Gly Leu ProAla Asn Lys Thr Val Ala Leu Gly Ser Asn Val Glu 260 265 270 Phe Met CysLys Val Tyr Ser Asp Pro Gln Pro His Ile Gln Trp Leu 275 280 285 Lys HisIle Glu Val Asn Gly Ser Lys Ile Gly Pro Asp Asn Leu Pro 290 295 300 TyrVal Gln Ile Leu Lys Thr Ala Gly Val Asn Thr Thr Asp Lys Glu 305 310 315320 Met Glu Val Leu His Leu Arg Asn Val Ser Phe Glu Asp Ala Gly Glu 325330 335 Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Leu Ser His His Ser Ala340 345 350 Trp Leu Thr Val Leu Glu Ala Leu Glu Glu Arg Pro Ala Val MetThr 355 360 365 Ser Pro Leu Tyr Leu Glu Ser Arg Gly Gly Leu Val Pro ArgGly Ser 370 375 380 Gly Ser Pro Gly Leu Gln Glu Pro Lys Ser Cys Asp LysThr His Thr 385 390 395 400 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe 405 410 415 Leu Phe Pro Pro Lys Pro Lys Asp Thr LeuMet Ile Ser Arg Thr Pro 420 425 430 Glu Val Thr Cys Val Val Val Asp ValSer His Glu Asp Pro Glu Val 435 440 445 Lys Phe Asn Trp Tyr Val Asp GlyVal Glu Val His Asn Ala Lys Thr 450 455 460 Lys Pro Arg Glu Glu Gln TyrAsn Ser Thr Tyr Arg Val Val Ser Val 465 470 475 480 Leu Thr Val Leu HisGln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 485 490 495 Lys Val Ser AsnLys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 500 505 510 Lys Ala LysGly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 515 520 525 Ser ArgAsp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 530 535 540 LysGly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 545 550 555560 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 565570 575 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp580 585 590 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala LeuHis 595 600 605 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys610 615 620 3 1578 DNA Homo sapiens CDS (1)...(1578) 3 atg tgg agc tggaag tgc ctc ctc ttc tgg gct gtg ctg gtc aca gcc 48 Met Trp Ser Trp LysCys Leu Leu Phe Trp Ala Val Leu Val Thr Ala 1 5 10 15 aca ctc tgc accgct agg ccg tcc ccg acc ttg cct gaa caa gat gct 96 Thr Leu Cys Thr AlaArg Pro Ser Pro Thr Leu Pro Glu Gln Asp Ala 20 25 30 ctc ccc tcc tcg gaggat gat gat gat gat gat gac tcc tct tca gag 144 Leu Pro Ser Ser Glu AspAsp Asp Asp Asp Asp Asp Ser Ser Ser Glu 35 40 45 gag aaa gaa aca gat aacacc aaa cca aac ccc gta gct cca tat tgg 192 Glu Lys Glu Thr Asp Asn ThrLys Pro Asn Pro Val Ala Pro Tyr Trp 50 55 60 aca tcc cca gaa aag atg gaaaag aaa ttg cat gca gtg ccg gct gcc 240 Thr Ser Pro Glu Lys Met Glu LysLys Leu His Ala Val Pro Ala Ala 65 70 75 80 aag aca gtg aag ttc aaa tgccct tcc agt ggg acc cca aac ccc aca 288 Lys Thr Val Lys Phe Lys Cys ProSer Ser Gly Thr Pro Asn Pro Thr 85 90 95 ctg cgc tgg ttg aaa aat ggc aaagaa ttc aaa cct gac cac aga att 336 Leu Arg Trp Leu Lys Asn Gly Lys GluPhe Lys Pro Asp His Arg Ile 100 105 110 gga ggc tac aag gtc cgt tat gccacc tgg agc atc ata atg gac tct 384 Gly Gly Tyr Lys Val Arg Tyr Ala ThrTrp Ser Ile Ile Met Asp Ser 115 120 125 gtg gtg ccc tct gac aag ggc aactac acc tgc att gtg gag aat gag 432 Val Val Pro Ser Asp Lys Gly Asn TyrThr Cys Ile Val Glu Asn Glu 130 135 140 tac ggc agc atc aac cac aca taccag ctg gat gtc gtg gag cgg tcc 480 Tyr Gly Ser Ile Asn His Thr Tyr GlnLeu Asp Val Val Glu Arg Ser 145 150 155 160 cct cac cgg ccc atc ctg caagca ggg ttg ccc gcc aac aaa aca gtg 528 Pro His Arg Pro Ile Leu Gln AlaGly Leu Pro Ala Asn Lys Thr Val 165 170 175 gcc ctg ggt agc aac gtg gagttc atg tgt aag gtg tac agt gac ccg 576 Ala Leu Gly Ser Asn Val Glu PheMet Cys Lys Val Tyr Ser Asp Pro 180 185 190 cag ccg cac atc cag tgg ctaaag cac atc gag gtg aat ggg agc aag 624 Gln Pro His Ile Gln Trp Leu LysHis Ile Glu Val Asn Gly Ser Lys 195 200 205 att ggc cca gac aac ctg ccttat gtc cag atc ttg aag act gct gga 672 Ile Gly Pro Asp Asn Leu Pro TyrVal Gln Ile Leu Lys Thr Ala Gly 210 215 220 gtt aat acc acc gac aaa gagatg gag gtg ctt cac tta aga aat gtc 720 Val Asn Thr Thr Asp Lys Glu MetGlu Val Leu His Leu Arg Asn Val 225 230 235 240 tcc ttt gag gac gca ggggag tat acg tgc ttg gcg ggt aac tct atc 768 Ser Phe Glu Asp Ala Gly GluTyr Thr Cys Leu Ala Gly Asn Ser Ile 245 250 255 gga ctc tcc cat cac tctgca tgg ttg acc gtt ctg gaa gcc ctg gaa 816 Gly Leu Ser His His Ser AlaTrp Leu Thr Val Leu Glu Ala Leu Glu 260 265 270 gag agg ccg gca gtg atgacc tcg ccc ctg tac ctg gag ggc agc ggt 864 Glu Arg Pro Ala Val Met ThrSer Pro Leu Tyr Leu Glu Gly Ser Gly 275 280 285 tcc ccc ggg ttg cag gagccc aaa tct tgt gac aaa act cac aca tgc 912 Ser Pro Gly Leu Gln Glu ProLys Ser Cys Asp Lys Thr His Thr Cys 290 295 300 cca ccg tgc cca gca cctgaa ctc ctg ggg gga ccg tca gtc ttc ctc 960 Pro Pro Cys Pro Ala Pro GluLeu Leu Gly Gly Pro Ser Val Phe Leu 305 310 315 320 ttc ccc cca aaa cccaag gac acc ctc atg atc tcc cgg acc cct gag 1008 Phe Pro Pro Lys Pro LysAsp Thr Leu Met Ile Ser Arg Thr Pro Glu 325 330 335 gtc aca tgc gtg gtggtg gac gtg agc cac gaa gac cct gag gtc aag 1056 Val Thr Cys Val Val ValAsp Val Ser His Glu Asp Pro Glu Val Lys 340 345 350 ttc aac tgg tac gtggac ggc gtg gag gtg cat aat gcc aag aca aag 1104 Phe Asn Trp Tyr Val AspGly Val Glu Val His Asn Ala Lys Thr Lys 355 360 365 ccg cgg gag gag cagtac aac agc acg tac cgt gtg gtc agc gtc ctc 1152 Pro Arg Glu Glu Gln TyrAsn Ser Thr Tyr Arg Val Val Ser Val Leu 370 375 380 acc gtc ctg cac caggac tgg ctg aat ggc aag gag tac aag tgc aag 1200 Thr Val Leu His Gln AspTrp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 385 390 395 400 gtc tcc aac aaagcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa 1248 Val Ser Asn Lys AlaLeu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 405 410 415 gcc aaa ggg cagccc cga gaa cca cag gtg tac acc ctg ccc cca tcc 1296 Ala Lys Gly Gln ProArg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 420 425 430 cgg gat gag ctgacc aag aac cag gtc agc ctg acc tgc ctg gtc aaa 1344 Arg Asp Glu Leu ThrLys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 435 440 445 ggc ttc tat cccagc gac atc gcc gtg gag tgg gag agc aat ggg cag 1392 Gly Phe Tyr Pro SerAsp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 450 455 460 ccg gag aac aactac aag acc acg cct ccc gtg ctg gac tcc gac ggc 1440 Pro Glu Asn Asn TyrLys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 465 470 475 480 tcc ttc ttcctc tac agc aag ctc acc gtg gac aag agc agg tgg cag 1488 Ser Phe Phe LeuTyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 485 490 495 cag ggg aacgtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac 1536 Gln Gly Asn ValPhe Ser Cys Ser Val Met His Glu Ala Leu His Asn 500 505 510 cac tac acgcag aag agc ctc tcc ctg tct ccg ggt aaa tga 1578 His Tyr Thr Gln Lys SerLeu Ser Leu Ser Pro Gly Lys * 515 520 525 4 525 PRT Homo sapiens 4 MetTrp Ser Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val Thr Ala 1 5 10 15Thr Leu Cys Thr Ala Arg Pro Ser Pro Thr Leu Pro Glu Gln Asp Ala 20 25 30Leu Pro Ser Ser Glu Asp Asp Asp Asp Asp Asp Asp Ser Ser Ser Glu 35 40 45Glu Lys Glu Thr Asp Asn Thr Lys Pro Asn Pro Val Ala Pro Tyr Trp 50 55 60Thr Ser Pro Glu Lys Met Glu Lys Lys Leu His Ala Val Pro Ala Ala 65 70 7580 Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr Pro Asn Pro Thr 85 9095 Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro Asp His Arg Ile 100105 110 Gly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser Ile Ile Met Asp Ser115 120 125 Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys Ile Val Glu AsnGlu 130 135 140 Tyr Gly Ser Ile Asn His Thr Tyr Gln Leu Asp Val Val GluArg Ser 145 150 155 160 Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro AlaAsn Lys Thr Val 165 170 175 Ala Leu Gly Ser Asn Val Glu Phe Met Cys LysVal Tyr Ser Asp Pro 180 185 190 Gln Pro His Ile Gln Trp Leu Lys His IleGlu Val Asn Gly Ser Lys 195 200 205 Ile Gly Pro Asp Asn Leu Pro Tyr ValGln Ile Leu Lys Thr Ala Gly 210 215 220 Val Asn Thr Thr Asp Lys Glu MetGlu Val Leu His Leu Arg Asn Val 225 230 235 240 Ser Phe Glu Asp Ala GlyGlu Tyr Thr Cys Leu Ala Gly Asn Ser Ile 245 250 255 Gly Leu Ser His HisSer Ala Trp Leu Thr Val Leu Glu Ala Leu Glu 260 265 270 Glu Arg Pro AlaVal Met Thr Ser Pro Leu Tyr Leu Glu Gly Ser Gly 275 280 285 Ser Pro GlyLeu Gln Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 290 295 300 Pro ProCys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 305 310 315 320Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 325 330335 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 340345 350 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys355 360 365 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser ValLeu 370 375 380 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr LysCys Lys 385 390 395 400 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu LysThr Ile Ser Lys 405 410 415 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val TyrThr Leu Pro Pro Ser 420 425 430 Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys 435 440 445 Gly Phe Tyr Pro Ser Asp Ile Ala ValGlu Trp Glu Ser Asn Gly Gln 450 455 460 Pro Glu Asn Asn Tyr Lys Thr ThrPro Pro Val Leu Asp Ser Asp Gly 465 470 475 480 Ser Phe Phe Leu Tyr SerLys Leu Thr Val Asp Lys Ser Arg Trp Gln 485 490 495 Gln Gly Asn Val PheSer Cys Ser Val Met His Glu Ala Leu His Asn 500 505 510 His Tyr Thr GlnLys Ser Leu Ser Leu Ser Pro Gly Lys 515 520 525 5 1494 DNA Homo sapiensCDS (1)...(1494) 5 atg tgg agc tgg aag tgc ctc ctc ttc tgg gct gtg ctggtc aca gcc 48 Met Trp Ser Trp Lys Cys Leu Leu Phe Trp Ala Val Leu ValThr Ala 1 5 10 15 aca ctc tgc acc gct agg ccg tcc ccg acc ttg cct gaacaa ccc gta 96 Thr Leu Cys Thr Ala Arg Pro Ser Pro Thr Leu Pro Glu GlnPro Val 20 25 30 gct cca tat tgg aca tcc cca gaa aag atg gaa aag aaa ttgcat gca 144 Ala Pro Tyr Trp Thr Ser Pro Glu Lys Met Glu Lys Lys Leu HisAla 35 40 45 gtg ccg gct gcc aag aca gtg aag ttc aaa tgc cct tcc agt gggacc 192 Val Pro Ala Ala Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr50 55 60 cca aac ccc aca ctg cgc tgg ttg aaa aat ggc aaa gaa ttc aaa cct240 Pro Asn Pro Thr Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro 6570 75 80 gac cac aga att ggt ggc tac aag gtc cgt tat gcc acc tgg agc atc288 Asp His Arg Ile Gly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser Ile 8590 95 ata atg gac tct gtg gtg ccc tct gac aag ggc aac tac acc tgc att336 Ile Met Asp Ser Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys Ile 100105 110 gtg gag aat gag tac ggc agc atc aac cac aca tac cag ctg gat gtc384 Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr Gln Leu Asp Val 115120 125 gtg gag cgg tcc cct cac cgg ccc atc ctg caa gca ggg ttg ccc gcc432 Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala 130135 140 aac aaa aca gtg gcc ctg ggt agc aac gtg gag ttc atg tgt aag gtg480 Asn Lys Thr Val Ala Leu Gly Ser Asn Val Glu Phe Met Cys Lys Val 145150 155 160 tac agt gac ccg cag ccg cac atc cag tgg cta aag cac atc gaggtg 528 Tyr Ser Asp Pro Gln Pro His Ile Gln Trp Leu Lys His Ile Glu Val165 170 175 aat ggg agc aag att ggc cca gac aac ctg cct tat gtc cag atcttg 576 Asn Gly Ser Lys Ile Gly Pro Asp Asn Leu Pro Tyr Val Gln Ile Leu180 185 190 aag act gct gga gtt aat acc acc gac aaa gag atg gag gtg cttcac 624 Lys Thr Ala Gly Val Asn Thr Thr Asp Lys Glu Met Glu Val Leu His195 200 205 tta aga aat gtc tcc ttt gag gac gca ggg gag tat acg tgc ttggcg 672 Leu Arg Asn Val Ser Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala210 215 220 ggt aac tct atc gga ctc tcc cat cac tct gca tgg ttg acc gttctg 720 Gly Asn Ser Ile Gly Leu Ser His His Ser Ala Trp Leu Thr Val Leu225 230 235 240 gaa gcc ctg gaa gag agg ccg gca gtg atg acc tcg ccc ctgtac ctg 768 Glu Ala Leu Glu Glu Arg Pro Ala Val Met Thr Ser Pro Leu TyrLeu 245 250 255 gag ggc agc ggt tcc ccc ggg ttg cag gag ccc aaa tct tgtgac aaa 816 Glu Gly Ser Gly Ser Pro Gly Leu Gln Glu Pro Lys Ser Cys AspLys 260 265 270 act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggggga ccg 864 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly GlyPro 275 280 285 tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atgatc tcc 912 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met IleSer 290 295 300 cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cacgaa gac 960 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His GluAsp 305 310 315 320 cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gaggtg cat aat 1008 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn 325 330 335 gcc aag aca aag ccg cgg gag gag cag tac aac agc acgtac cgt gtg 1056 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val 340 345 350 gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aatggc aag gag 1104 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu 355 360 365 tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc cccatc gag aaa 1152 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys 370 375 380 acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca caggtg tac acc 1200 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr 385 390 395 400 ctg ccc cca tcc cgg gat gag ctg acc aag aac caggtc agc ctg acc 1248 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln ValSer Leu Thr 405 410 415 tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gccgtg gag tgg gag 1296 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala ValGlu Trp Glu 420 425 430 agc aat ggg cag ccg gag aac aac tac aag acc acgcct ccc gtg ctg 1344 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr ProPro Val Leu 435 440 445 gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctcacc gtg gac aag 1392 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu ThrVal Asp Lys 450 455 460 agc agg tgg cag cag ggg aac gtc ttc tca tgc tccgtg atg cat gag 1440 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser ValMet His Glu 465 470 475 480 gct ctg cac aac cac tac acg cag aag agc ctctcc ctg tct ccg ggt 1488 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu SerLeu Ser Pro Gly 485 490 495 aaa tga 1494 Lys * 6 497 PRT Homo sapiens 6Met Trp Ser Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val Thr Ala 1 5 1015 Thr Leu Cys Thr Ala Arg Pro Ser Pro Thr Leu Pro Glu Gln Pro Val 20 2530 Ala Pro Tyr Trp Thr Ser Pro Glu Lys Met Glu Lys Lys Leu His Ala 35 4045 Val Pro Ala Ala Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr 50 5560 Pro Asn Pro Thr Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro 65 7075 80 Asp His Arg Ile Gly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser Ile 8590 95 Ile Met Asp Ser Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys Ile100 105 110 Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr Gln Leu AspVal 115 120 125 Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly LeuPro Ala 130 135 140 Asn Lys Thr Val Ala Leu Gly Ser Asn Val Glu Phe MetCys Lys Val 145 150 155 160 Tyr Ser Asp Pro Gln Pro His Ile Gln Trp LeuLys His Ile Glu Val 165 170 175 Asn Gly Ser Lys Ile Gly Pro Asp Asn LeuPro Tyr Val Gln Ile Leu 180 185 190 Lys Thr Ala Gly Val Asn Thr Thr AspLys Glu Met Glu Val Leu His 195 200 205 Leu Arg Asn Val Ser Phe Glu AspAla Gly Glu Tyr Thr Cys Leu Ala 210 215 220 Gly Asn Ser Ile Gly Leu SerHis His Ser Ala Trp Leu Thr Val Leu 225 230 235 240 Glu Ala Leu Glu GluArg Pro Ala Val Met Thr Ser Pro Leu Tyr Leu 245 250 255 Glu Gly Ser GlySer Pro Gly Leu Gln Glu Pro Lys Ser Cys Asp Lys 260 265 270 Thr His ThrCys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 275 280 285 Ser ValPhe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 290 295 300 ArgThr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 305 310 315320 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 325330 335 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val340 345 350 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly LysGlu 355 360 365 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys 370 375 380 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro GlnVal Tyr Thr 385 390 395 400 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys AsnGln Val Ser Leu Thr 405 410 415 Cys Leu Val Lys Gly Phe Tyr Pro Ser AspIle Ala Val Glu Trp Glu 420 425 430 Ser Asn Gly Gln Pro Glu Asn Asn TyrLys Thr Thr Pro Pro Val Leu 435 440 445 Asp Ser Asp Gly Ser Phe Phe LeuTyr Ser Lys Leu Thr Val Asp Lys 450 455 460 Ser Arg Trp Gln Gln Gly AsnVal Phe Ser Cys Ser Val Met His Glu 465 470 475 480 Ala Leu His Asn HisTyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 485 490 495 Lys 7 1578 DNAHomo sapiens CDS (1)...(1578) 7 atg tgg agc tgg aag tgc ctc ctc ttc tgggct gtg ctg gtc aca gcc 48 Met Trp Ser Trp Lys Cys Leu Leu Phe Trp AlaVal Leu Val Thr Ala 1 5 10 15 aca ctc tgc acc gct agg ccg tcc ccg accttg cct gaa caa gat gct 96 Thr Leu Cys Thr Ala Arg Pro Ser Pro Thr LeuPro Glu Gln Asp Ala 20 25 30 ctc ccc tcc tcg gag gat gat gat gat gat gatgac tcc tct tca gag 144 Leu Pro Ser Ser Glu Asp Asp Asp Asp Asp Asp AspSer Ser Ser Glu 35 40 45 gag aaa gaa aca gat aac acc aaa cca aac ccc gtagct cca tat tgg 192 Glu Lys Glu Thr Asp Asn Thr Lys Pro Asn Pro Val AlaPro Tyr Trp 50 55 60 aca tcc cca gaa aag atg gaa aag aaa ttg cat gca gtgccg gct gcc 240 Thr Ser Pro Glu Lys Met Glu Lys Lys Leu His Ala Val ProAla Ala 65 70 75 80 aag aca gtg aag ttc aaa tgc cct tcc agt ggg acc ccaaac ccc aca 288 Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr Pro AsnPro Thr 85 90 95 ctg cgc tgg ttg aaa aat ggc aaa gaa ttc aaa cct gac cacaga att 336 Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro Asp His ArgIle 100 105 110 gga ggc tac aag gtc cgt tat gcc acc tgg agc atc ata atggac tct 384 Gly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser Ile Ile Met AspSer 115 120 125 gtg gtg ccc tct gac aag ggc aac tac acc tgc att gtg gagaat gag 432 Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys Ile Val Glu AsnGlu 130 135 140 tac ggc agc atc aac cac aca tac cag ctg gat gtc gtg gagcgg tcc 480 Tyr Gly Ser Ile Asn His Thr Tyr Gln Leu Asp Val Val Glu ArgSer 145 150 155 160 cct cac cgg ccc atc ctg caa gca ggg ttg ccc gcc aacaaa aca gtg 528 Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn LysThr Val 165 170 175 gcc ctg ggt agc aac gtg gag ttc atg tgt aag gtg tacagt gac ccg 576 Ala Leu Gly Ser Asn Val Glu Phe Met Cys Lys Val Tyr SerAsp Pro 180 185 190 cag ccg cac atc cag tgg cta aag cac atc gag gtg aatggg agc aag 624 Gln Pro His Ile Gln Trp Leu Lys His Ile Glu Val Asn GlySer Lys 195 200 205 att ggc cca gac aac ctg cct tat gtc cag atc ttg aagact gct gga 672 Ile Gly Pro Asp Asn Leu Pro Tyr Val Gln Ile Leu Lys ThrAla Gly 210 215 220 gtt aat acc acc gac aaa gag atg gag gtg ctt cac ttaaga aat gtc 720 Val Asn Thr Thr Asp Lys Glu Met Glu Val Leu His Leu ArgAsn Val 225 230 235 240 tcc ttt gag gac gca ggg gag tat acg tgc ttg gcgggt aac tct atc 768 Ser Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala GlyAsn Ser Ile 245 250 255 gga ctc tcc cat cac tct gca tgg ttg acc gtt ctggaa gcc ctg gaa 816 Gly Leu Ser His His Ser Ala Trp Leu Thr Val Leu GluAla Leu Glu 260 265 270 gag agg ccg gca gtg atg acc tcg ccc ctg tac ctggag ggc agc ggt 864 Glu Arg Pro Ala Val Met Thr Ser Pro Leu Tyr Leu GluGly Ser Gly 275 280 285 tcc ccc ggg ttg cag gag ccc aaa tct tgt gac aaaact cac aca tgc 912 Ser Pro Gly Leu Gln Glu Pro Lys Ser Cys Asp Lys ThrHis Thr Cys 290 295 300 cca ccg tgc cca gca cct gaa ctc gag ggg gga ccgtca gtc ttc ctc 960 Pro Pro Cys Pro Ala Pro Glu Leu Glu Gly Gly Pro SerVal Phe Leu 305 310 315 320 ttc ccc cca aaa ccc aag gac acc ctc atg atctcc cgg acc cct gag 1008 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile SerArg Thr Pro Glu 325 330 335 gtc aca tgc gtg gtg gtg gac gtg agc cac gaagac cct gag gtc aag 1056 Val Thr Cys Val Val Val Asp Val Ser His Glu AspPro Glu Val Lys 340 345 350 ttc aac tgg tac gtg gac ggc gtg gag gtg cataat gcc aag aca aag 1104 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His AsnAla Lys Thr Lys 355 360 365 ccg cgg gag gag cag tac aac agc acg tac cgtgtg gtc agc gtc ctc 1152 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg ValVal Ser Val Leu 370 375 380 acc gtc ctg cac cag gac tgg ctg aat ggc aaggag tac aag tgc aag 1200 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys GluTyr Lys Cys Lys 385 390 395 400 gtc tcc aac aaa gcc ctc cca gcc tcc atcgag aaa acc atc tcc aaa 1248 Val Ser Asn Lys Ala Leu Pro Ala Ser Ile GluLys Thr Ile Ser Lys 405 410 415 gcc aaa ggg cag ccc cga gaa cca cag gtgtac acc ctg ccc cca tcc 1296 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val TyrThr Leu Pro Pro Ser 420 425 430 cgg gat gag ctg acc aag aac cag gtc agcctg acc tgc ctg gtc aaa 1344 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser LeuThr Cys Leu Val Lys 435 440 445 ggc ttc tat ccc agc gac atc gcc gtg gagtgg gag agc aat ggg cag 1392 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu TrpGlu Ser Asn Gly Gln 450 455 460 ccg gag aac aac tac aag acc acg cct cccgtg ctg gac tcc gac ggc 1440 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro ValLeu Asp Ser Asp Gly 465 470 475 480 tcc ttc ttc ctc tac agc aag ctc accgtg gac aag agc agg tgg cag 1488 Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln 485 490 495 cag ggg aac gtc ttc tca tgc tcc gtgatg cat gag gct ctg cac aac 1536 Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn 500 505 510 cac tac acg cag aag agc ctc tcc ctgtct ccg ggt aaa tga 1578 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyLys * 515 520 525 8 525 PRT Homo sapiens 8 Met Trp Ser Trp Lys Cys LeuLeu Phe Trp Ala Val Leu Val Thr Ala 1 5 10 15 Thr Leu Cys Thr Ala ArgPro Ser Pro Thr Leu Pro Glu Gln Asp Ala 20 25 30 Leu Pro Ser Ser Glu AspAsp Asp Asp Asp Asp Asp Ser Ser Ser Glu 35 40 45 Glu Lys Glu Thr Asp AsnThr Lys Pro Asn Pro Val Ala Pro Tyr Trp 50 55 60 Thr Ser Pro Glu Lys MetGlu Lys Lys Leu His Ala Val Pro Ala Ala 65 70 75 80 Lys Thr Val Lys PheLys Cys Pro Ser Ser Gly Thr Pro Asn Pro Thr 85 90 95 Leu Arg Trp Leu LysAsn Gly Lys Glu Phe Lys Pro Asp His Arg Ile 100 105 110 Gly Gly Tyr LysVal Arg Tyr Ala Thr Trp Ser Ile Ile Met Asp Ser 115 120 125 Val Val ProSer Asp Lys Gly Asn Tyr Thr Cys Ile Val Glu Asn Glu 130 135 140 Tyr GlySer Ile Asn His Thr Tyr Gln Leu Asp Val Val Glu Arg Ser 145 150 155 160Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn Lys Thr Val 165 170175 Ala Leu Gly Ser Asn Val Glu Phe Met Cys Lys Val Tyr Ser Asp Pro 180185 190 Gln Pro His Ile Gln Trp Leu Lys His Ile Glu Val Asn Gly Ser Lys195 200 205 Ile Gly Pro Asp Asn Leu Pro Tyr Val Gln Ile Leu Lys Thr AlaGly 210 215 220 Val Asn Thr Thr Asp Lys Glu Met Glu Val Leu His Leu ArgAsn Val 225 230 235 240 Ser Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu AlaGly Asn Ser Ile 245 250 255 Gly Leu Ser His His Ser Ala Trp Leu Thr ValLeu Glu Ala Leu Glu 260 265 270 Glu Arg Pro Ala Val Met Thr Ser Pro LeuTyr Leu Glu Gly Ser Gly 275 280 285 Ser Pro Gly Leu Gln Glu Pro Lys SerCys Asp Lys Thr His Thr Cys 290 295 300 Pro Pro Cys Pro Ala Pro Glu LeuGlu Gly Gly Pro Ser Val Phe Leu 305 310 315 320 Phe Pro Pro Lys Pro LysAsp Thr Leu Met Ile Ser Arg Thr Pro Glu 325 330 335 Val Thr Cys Val ValVal Asp Val Ser His Glu Asp Pro Glu Val Lys 340 345 350 Phe Asn Trp TyrVal Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 355 360 365 Pro Arg GluGlu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 370 375 380 Thr ValLeu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 385 390 395 400Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys 405 410415 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 420425 430 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys435 440 445 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn GlyGln 450 455 460 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp SerAsp Gly 465 470 475 480 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp LysSer Arg Trp Gln 485 490 495 Gln Gly Asn Val Phe Ser Cys Ser Val Met HisGlu Ala Leu His Asn 500 505 510 His Tyr Thr Gln Lys Ser Leu Ser Leu SerPro Gly Lys 515 520 525 9 1494 DNA Homo sapiens CDS (1)...(1494) 9 atgtgg agc tgg aag tgc ctc ctc ttc tgg gct gtg ctg gtc aca gcc 48 Met TrpSer Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val Thr Ala 1 5 10 15 acactc tgc acc gct agg ccg tcc ccg acc ttg cct gaa caa ccc gta 96 Thr LeuCys Thr Ala Arg Pro Ser Pro Thr Leu Pro Glu Gln Pro Val 20 25 30 gct ccatat tgg aca tcc cca gaa aag atg gaa aag aaa ttg cat gca 144 Ala Pro TyrTrp Thr Ser Pro Glu Lys Met Glu Lys Lys Leu His Ala 35 40 45 gtg ccg gctgcc aag aca gtg aag ttc aaa tgc cct tcc agt ggg acc 192 Val Pro Ala AlaLys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr 50 55 60 cca aac ccc acactg cgc tgg ttg aaa aat ggc aaa gaa ttc aaa cct 240 Pro Asn Pro Thr LeuArg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro 65 70 75 80 gac cac aga attggt ggc tac aag gtc cgt tat gcc acc tgg agc atc 288 Asp His Arg Ile GlyGly Tyr Lys Val Arg Tyr Ala Thr Trp Ser Ile 85 90 95 ata atg gac tct gtggtg ccc tct gac aag ggc aac tac acc tgc att 336 Ile Met Asp Ser Val ValPro Ser Asp Lys Gly Asn Tyr Thr Cys Ile 100 105 110 gtg gag aat gag tacggc agc atc aac cac aca tac cag ctg gat gtc 384 Val Glu Asn Glu Tyr GlySer Ile Asn His Thr Tyr Gln Leu Asp Val 115 120 125 gtg gag cgg tcc cctcac cgg ccc atc ctg caa gca ggg ttg ccc gcc 432 Val Glu Arg Ser Pro HisArg Pro Ile Leu Gln Ala Gly Leu Pro Ala 130 135 140 aac aaa aca gtg gccctg ggt agc aac gtg gag ttc atg tgt aag gtg 480 Asn Lys Thr Val Ala LeuGly Ser Asn Val Glu Phe Met Cys Lys Val 145 150 155 160 tac agt gac ccgcag ccg cac atc cag tgg cta aag cac atc gag gtg 528 Tyr Ser Asp Pro GlnPro His Ile Gln Trp Leu Lys His Ile Glu Val 165 170 175 aat ggg agc aagatt ggc cca gac aac ctg cct tat gtc cag atc ttg 576 Asn Gly Ser Lys IleGly Pro Asp Asn Leu Pro Tyr Val Gln Ile Leu 180 185 190 aag act gct ggagtt aat acc acc gac aaa gag atg gag gtg ctt cac 624 Lys Thr Ala Gly ValAsn Thr Thr Asp Lys Glu Met Glu Val Leu His 195 200 205 tta aga aat gtctcc ttt gag gac gca ggg gag tat acg tgc ttg gcg 672 Leu Arg Asn Val SerPhe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala 210 215 220 ggt aac tct atcgga ctc tcc cat cac tct gca tgg ttg acc gtt ctg 720 Gly Asn Ser Ile GlyLeu Ser His His Ser Ala Trp Leu Thr Val Leu 225 230 235 240 gaa gcc ctggaa gag agg ccg gca gtg atg acc tcg ccc ctg tac ctg 768 Glu Ala Leu GluGlu Arg Pro Ala Val Met Thr Ser Pro Leu Tyr Leu 245 250 255 gag ggc agcggt tcc ccc ggg ttg cag gag ccc aaa tct tgt gac aaa 816 Glu Gly Ser GlySer Pro Gly Leu Gln Glu Pro Lys Ser Cys Asp Lys 260 265 270 act cac acatgc cca ccg tgc cca gca cct gaa ctc gag ggg gga ccg 864 Thr His Thr CysPro Pro Cys Pro Ala Pro Glu Leu Glu Gly Gly Pro 275 280 285 tca gtc ttcctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc 912 Ser Val Phe LeuPhe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 290 295 300 cgg acc cctgag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac 960 Arg Thr Pro GluVal Thr Cys Val Val Val Asp Val Ser His Glu Asp 305 310 315 320 cct gaggtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat 1008 Pro Glu ValLys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 325 330 335 gcc aagaca aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg 1056 Ala Lys ThrLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 340 345 350 gtc agcgtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag 1104 Val Ser ValLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 355 360 365 tac aagtgc aag gtc tcc aac aaa gcc ctc cca gcc tcc atc gag aaa 1152 Tyr Lys CysLys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys 370 375 380 acc atctcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc 1200 Thr Ile SerLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 385 390 395 400 ctgccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc 1248 Leu ProPro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 405 410 415 tgcctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag 1296 Cys LeuVal Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 420 425 430 agcaat ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg 1344 Ser AsnGly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 435 440 445 gactcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag 1392 Asp SerAsp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 450 455 460 agcagg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag 1440 Ser ArgTrp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 465 470 475 480gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt 1488 AlaLeu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 485 490 495aaa tga 1494 Lys * 10 497 PRT Homo sapiens 10 Met Trp Ser Trp Lys CysLeu Leu Phe Trp Ala Val Leu Val Thr Ala 1 5 10 15 Thr Leu Cys Thr AlaArg Pro Ser Pro Thr Leu Pro Glu Gln Pro Val 20 25 30 Ala Pro Tyr Trp ThrSer Pro Glu Lys Met Glu Lys Lys Leu His Ala 35 40 45 Val Pro Ala Ala LysThr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr 50 55 60 Pro Asn Pro Thr LeuArg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro 65 70 75 80 Asp His Arg IleGly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser Ile 85 90 95 Ile Met Asp SerVal Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys Ile 100 105 110 Val Glu AsnGlu Tyr Gly Ser Ile Asn His Thr Tyr Gln Leu Asp Val 115 120 125 Val GluArg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala 130 135 140 AsnLys Thr Val Ala Leu Gly Ser Asn Val Glu Phe Met Cys Lys Val 145 150 155160 Tyr Ser Asp Pro Gln Pro His Ile Gln Trp Leu Lys His Ile Glu Val 165170 175 Asn Gly Ser Lys Ile Gly Pro Asp Asn Leu Pro Tyr Val Gln Ile Leu180 185 190 Lys Thr Ala Gly Val Asn Thr Thr Asp Lys Glu Met Glu Val LeuHis 195 200 205 Leu Arg Asn Val Ser Phe Glu Asp Ala Gly Glu Tyr Thr CysLeu Ala 210 215 220 Gly Asn Ser Ile Gly Leu Ser His His Ser Ala Trp LeuThr Val Leu 225 230 235 240 Glu Ala Leu Glu Glu Arg Pro Ala Val Met ThrSer Pro Leu Tyr Leu 245 250 255 Glu Gly Ser Gly Ser Pro Gly Leu Gln GluPro Lys Ser Cys Asp Lys 260 265 270 Thr His Thr Cys Pro Pro Cys Pro AlaPro Glu Leu Glu Gly Gly Pro 275 280 285 Ser Val Phe Leu Phe Pro Pro LysPro Lys Asp Thr Leu Met Ile Ser 290 295 300 Arg Thr Pro Glu Val Thr CysVal Val Val Asp Val Ser His Glu Asp 305 310 315 320 Pro Glu Val Lys PheAsn Trp Tyr Val Asp Gly Val Glu Val His Asn 325 330 335 Ala Lys Thr LysPro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 340 345 350 Val Ser ValLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 355 360 365 Tyr LysCys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys 370 375 380 ThrIle Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 385 390 395400 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 405410 415 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu420 425 430 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro ValLeu 435 440 445 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys 450 455 460 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser ValMet His Glu 465 470 475 480 Ala Leu His Asn His Tyr Thr Gln Lys Ser LeuSer Leu Ser Pro Gly 485 490 495 Lys 11 1467 DNA Homo sapiens CDS(1)...(1467) 11 atg tgg agc tgg aag tgc ctc ctc ttc tgg gct gtg ctg gtcaca gcc 48 Met Trp Ser Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val ThrAla 1 5 10 15 aca ctc tgc acc gct agg ccg tcc ccg acc ttg cct gaa caaccc gta 96 Thr Leu Cys Thr Ala Arg Pro Ser Pro Thr Leu Pro Glu Gln ProVal 20 25 30 gct cca tat tgg aca tcc cca gaa aag atg gaa aag aaa ttg catgca 144 Ala Pro Tyr Trp Thr Ser Pro Glu Lys Met Glu Lys Lys Leu His Ala35 40 45 gtg ccg gct gcc aag aca gtg aag ttc aaa tgc cct tcc agt ggg acc192 Val Pro Ala Ala Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr 5055 60 cca aac ccc aca ctg cgc tgg ttg aaa aat ggc aaa gaa ttc aaa cct240 Pro Asn Pro Thr Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro 6570 75 80 gac cac aga att ggt ggc tac aag gtc cgt tat gcc acc tgg agc atc288 Asp His Arg Ile Gly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser Ile 8590 95 ata atg gac tct gtg gtg ccc tct gac aag ggc aac tac acc tgc att336 Ile Met Asp Ser Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys Ile 100105 110 gtg gag aat gag tac ggc agc atc aac cac aca tac cag ctg gat gtc384 Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr Gln Leu Asp Val 115120 125 gtg gag cgg tcc cct cac cgg ccc atc ctg caa gca ggg ttg ccc gcc432 Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala 130135 140 aac aaa aca gtg gcc ctg ggt agc aac gtg gag ttc atg tgt aag gtg480 Asn Lys Thr Val Ala Leu Gly Ser Asn Val Glu Phe Met Cys Lys Val 145150 155 160 tac agt gac ccg cag ccg cac atc cag tgg cta aag cac atc gaggtg 528 Tyr Ser Asp Pro Gln Pro His Ile Gln Trp Leu Lys His Ile Glu Val165 170 175 aat ggg agc aag att ggc cca gac aac ctg cct tat gtc cag atcttg 576 Asn Gly Ser Lys Ile Gly Pro Asp Asn Leu Pro Tyr Val Gln Ile Leu180 185 190 aag act gct gga gtt aat acc acc gac aaa gag atg gag gtg cttcac 624 Lys Thr Ala Gly Val Asn Thr Thr Asp Lys Glu Met Glu Val Leu His195 200 205 tta aga aat gtc tcc ttt gag gac gca ggg gag tat acg tgc ttggcg 672 Leu Arg Asn Val Ser Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala210 215 220 ggt aac tct atc gga ctc tcc cat cac tct gca tgg ttg acc gttctg 720 Gly Asn Ser Ile Gly Leu Ser His His Ser Ala Trp Leu Thr Val Leu225 230 235 240 gaa gcc ctg gaa gag agg ccg gca gtg atg acc tcg ccc ctgtac ctg 768 Glu Ala Leu Glu Glu Arg Pro Ala Val Met Thr Ser Pro Leu TyrLeu 245 250 255 gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg tgccca gca 816 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys ProAla 260 265 270 cct gaa ctc gag ggg gga ccg tca gtc ttc ctc ttc ccc ccaaaa ccc 864 Pro Glu Leu Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro LysPro 275 280 285 aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgcgtg gtg 912 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys ValVal 290 295 300 gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tggtac gtg 960 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp TyrVal 305 310 315 320 gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgggag gag cag 1008 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg GluGlu Gln 325 330 335 tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtcctg cac cag 1056 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val LeuHis Gln 340 345 350 gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tccaac aaa gcc 1104 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser AsnLys Ala 355 360 365 ctc cca gcc tcc atc gag aaa acc atc tcc aaa gcc aaaggg cag ccc 1152 Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln Pro 370 375 380 cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gatgag ctg acc 1200 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp GluLeu Thr 385 390 395 400 aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggcttc tat ccc agc 1248 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly PheTyr Pro Ser 405 410 415 gac atc gcc gtg gag tgg gag agc aat ggg cag ccggag aac aac tac 1296 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro GluAsn Asn Tyr 420 425 430 aag acc acg cct ccc gtg ctg gac tcc gac ggc tccttc ttc ctc tac 1344 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser PhePhe Leu Tyr 435 440 445 agc aag ctc acc gtg gac aag agc agg tgg cag cagggg aac gtc ttc 1392 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln GlyAsn Val Phe 450 455 460 tca tgc tcc gtg atg cat gag gct ctg cac aac cactac acg cag aag 1440 Ser Cys Ser Val Met His Glu Ala Leu His Asn His TyrThr Gln Lys 465 470 475 480 agc ctc tcc ctg tct ccg ggt aaa tga 1467 SerLeu Ser Leu Ser Pro Gly Lys * 485 12 488 PRT Homo sapiens 12 Met Trp SerTrp Lys Cys Leu Leu Phe Trp Ala Val Leu Val Thr Ala 1 5 10 15 Thr LeuCys Thr Ala Arg Pro Ser Pro Thr Leu Pro Glu Gln Pro Val 20 25 30 Ala ProTyr Trp Thr Ser Pro Glu Lys Met Glu Lys Lys Leu His Ala 35 40 45 Val ProAla Ala Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr 50 55 60 Pro AsnPro Thr Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro 65 70 75 80 AspHis Arg Ile Gly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser Ile 85 90 95 IleMet Asp Ser Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys Ile 100 105 110Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr Gln Leu Asp Val 115 120125 Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala 130135 140 Asn Lys Thr Val Ala Leu Gly Ser Asn Val Glu Phe Met Cys Lys Val145 150 155 160 Tyr Ser Asp Pro Gln Pro His Ile Gln Trp Leu Lys His IleGlu Val 165 170 175 Asn Gly Ser Lys Ile Gly Pro Asp Asn Leu Pro Tyr ValGln Ile Leu 180 185 190 Lys Thr Ala Gly Val Asn Thr Thr Asp Lys Glu MetGlu Val Leu His 195 200 205 Leu Arg Asn Val Ser Phe Glu Asp Ala Gly GluTyr Thr Cys Leu Ala 210 215 220 Gly Asn Ser Ile Gly Leu Ser His His SerAla Trp Leu Thr Val Leu 225 230 235 240 Glu Ala Leu Glu Glu Arg Pro AlaVal Met Thr Ser Pro Leu Tyr Leu 245 250 255 Glu Pro Lys Ser Cys Asp LysThr His Thr Cys Pro Pro Cys Pro Ala 260 265 270 Pro Glu Leu Glu Gly GlyPro Ser Val Phe Leu Phe Pro Pro Lys Pro 275 280 285 Lys Asp Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val 290 295 300 Val Asp Val SerHis Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 305 310 315 320 Asp GlyVal Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 325 330 335 TyrAsn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 340 345 350Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 355 360365 Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 370375 380 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr385 390 395 400 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe TyrPro Ser 405 410 415 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro GluAsn Asn Tyr 420 425 430 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly SerPhe Phe Leu Tyr 435 440 445 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp GlnGln Gly Asn Val Phe 450 455 460 Ser Cys Ser Val Met His Glu Ala Leu HisAsn His Tyr Thr Gln Lys 465 470 475 480 Ser Leu Ser Leu Ser Pro Gly Lys485

That which is claimed:
 1. A polypeptide comprising a fibroblast growthfactor (FGF) receptor extracellular domain fused to a heterologousoligomerization domain wherein said extracellular domain lacks the Ig Isegment, and wherein said heterologous oligomerization domain isselected from the group consisting of a) the Fc region of animmunoglobulin molecule; b) the hinge region of an immunoglobulinmolecule; c) the CH1 region of an immunoglobulin molecule; d) the CH2region of an immunoglobulin molecule; e) the CH3 region of animmunoglobulin molecule; f) the CH4 region of an immunoglobulinmolecule; g) the light chain of an immunoglobulin molecule; and h) apeptide comprising a leucine zipper motif.
 2. The polypeptide of claim1, wherein said oligomerization domain is capable of forming an oligomerwhich is at least a dimer with at least one other polypeptide comprisingsaid oligomerization domain of claim
 1. 3. The polypeptide of claim 2,wherein said oligomer is a homodimer.
 4. The polypeptide of claim 2,wherein said oligomer is a heterodimer.
 5. The polypeptide of claim 1,wherein said polypeptide is capable of binding FGF.
 6. The polypeptideof claim 1 wherein said immonoglobulin is selected from the group IgG,IgE, IgA, IgD, and IgM.
 7. The polypeptide of claim 6, wherein saidimmunoglobulin is human immunoglobulin.
 8. The polypeptide of claim 1,wherein said oligomerization domain is the Fc region of animmunoglobulin molecule, and wherein the hinge region contained in saidFc region is capable of self dimerization, and wherein the CH2 and CH3regions contained in said Fc region are capable of increasing thecirculating half-life of said polypeptide, and wherein said polypeptideis capable of binding FGF.
 9. The polypeptide of claim 1 lacking theacid box segment of said extracellular domain.
 10. The polypeptide ofclaim 1 further comprising a variant of the Ig III segment of saidextracellular domain.
 11. The polypeptide of claim 1 lacking the acidbox segment of said extracellular domain and further comprising avariant of the Ig III segment of said extracellular domain.
 12. Thepolypeptide of claim 1, wherein said FGF receptor is selected from thegroup consisting of FGFR1, FGFR2, FGFR3 and FGFR4.
 13. The polypeptideof claim 1, wherein said FGF receptor is human FGFR1.
 14. A polypeptidehaving the amino acid sequence selected from the group consisting of: a)an amino acid sequence set forth in SEQ ID NO:4, 6, 8, 10, or 12; b) anamino acid sequence having at least 75% identity to the amino acidsequence set forth in SEQ ID NO:4, 6, 8, 10, or 12; and c) an amino acidsequence having at least 95% identity to the amino acid sequence setforth in SEQ ID NO:4, 6, 8, 10, or
 12. 15. A polynucleotide constructencoding said amino acid sequence of claim
 14. 16. A polynucleotideconstruct having the sequence selected from the group consisting of: a)the nucleotide sequence set forth in SEQ ID NO:3, 5, 7, 9, or 11; b) anucleotide sequence having at least 75% identity to the nucleotidesequence set forth in SEQ ID NO:3, 5, 7, 9, or 11; and c) a nucleotidesequence having at least 95% identity to the nucleotide sequence setforth in SEQ ID NO:3, 5, 7, 9, or
 11. 17. A polynucleotide constructencoding the polypeptide of claim
 1. 18. A viral vector comprising thepolynucleotide construct of claim
 17. 19. An isolated host cellcontaining or capable of expressing the polynucleotide construct ofclaim
 17. 20. A pharmacological composition comprising the vector ofclaim
 19. 21. A pharmacological composition comprising the polypeptideof claim
 1. 22. A method for treating a fibroblast growth factor(FGF)-mediated disorder in a mammal, said method comprisingadministering to said mammal a therapeutically effective amount of apolypeptide comprising an FGF receptor extracellular domain fused to aheterologous oligomerization domain, wherein said heterologousoligomerization domain is selected from the group consisting of a) theFc region of an immunoglobulin molecule; b) the hinge region of animmunoglobulin molecule; c) the CH1 region of an immunoglobulinmolecule; d) the CH2 region of an immunoglobulin molecule; e) the CH3region of an immunoglobulin molecule; f) the CH4 region of animmunoglobulin molecule; g) the light chain of an immunoglobulinmolecule; h) a peptide comprising a leucine zipper motif; and whereinsaid oligomerization domain is capable of forming an oligomer which isat least a dimer with at least one other polypeptide comprising saidoligomerization domain.
 23. The method of claim 22, wherein said mammalis human and wherein said FGF receptor extracellular domain and saidoligomerization domain are human polypeptides.
 24. The method of claim22, wherein said polypeptide has the amino acid sequence selected fromthe group consisting of: a) the amino acid sequence set forth in SEQ IDNO:2, 4, 6, 8, 10, or 12; b) an amino acid sequence having at least 75%identity to the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8,10, or 12; c) an amino acid sequence having at least 95% identity to theamino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, or
 12. 25.The method of claim 22, wherein said polypeptide has the amino acidsequence encoded by a polynucleotide having the sequence selected fromthe group consisting of: a) the nucleotide sequence set forth in SEQ IDNO:1, 3, 5, 7, 9, or 11; b) a nucleotide sequence having at least 75%identity to the nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 7,9, or 11; and c) a nucleotide sequence having at least 95% identity tothe nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, or
 11. 26.A method for treating a fibroblast growth factor (FGF)-mediated disorderin a mammal, said method comprising administering to said mammal atherapeutically effective amount of a polynucleotide comprising a codingregion encoding a polypeptide comprising an FGF receptor extracellulardomain fused to a heterologous oligomerization domain, wherein saidheterologous oligomerization domain is selected from the groupconsisting of a) the Fc region of an immunoglobulin molecule; b) thehinge region of an immunoglobulin molecule; c) the CH1 region of animmunoglobulin molecule; d) the CH2 region of an immunoglobulinmolecule; e) the CH3 region of an immunoglobulin molecule; f) the CH4region of an immunoglobulin molecule; g) the light chain of animmunoglobulin molecule; h) a peptide comprising a leucine zipper motif;and wherein said oligomerization domain is capable of forming anoligomer which is at least a dimer with at least one other polypeptidecomprising said oligomerization domain.
 27. The method of claim 26,wherein said polynucleotide has the sequence selected from the groupconsisting of: a) the nucleotide sequence set forth in SEQ ID NO:1, 3,5, 7, 9, or 11; b) a nucleotide sequence having at least 75% identity tothe nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, or 11; andc) a nucleotide sequence having at least 95% identity to the nucleotidesequence set forth in SEQ ID NO:1, 3, 5, 7, 9, or
 11. 28. A method fortreating an angiogenesis-mediated disorder in a mammal, said methodcomprising administering to said mammal a therapeutically effectiveamount of a polypeptide comprising a fibroblast growth factor (FGF)receptor extracellular domain fused to a heterologous oligomerizationdomain, wherein said heterologous oligomerization domain is selectedfrom the group consisting of: a) the Fc region of an immunoglobulinmolecule; b) the hinge region of an immunoglobulin molecule; c) the CH1region of an immunoglobulin molecule; d) the CH2 region of animmunoglobulin molecule; e) the CH3 region of an immunoglobulinmolecule; f) the CH4 region of an immunoglobulin molecule; g) the lightchain of an immunoglobulin molecule; h) a peptide comprising a leucinezipper motif; and wherein said oligomerization domain is capable offorming an oligomer which is at least a dimer with at least one otherpolypeptide comprising said oligomerization domain.
 29. A method fortreating an angiogenesis-mediated disorder in a mammal, said methodcomprising administering to said mammal a therapeutically effectiveamount of a polynucleotide comprising a coding region encoding apolypeptide comprising a fibroblast growth factor (FGF) receptorextracellular domain fused to a heterologous oligomerization domain,wherein said heterologous oligomerization domain is selected from thegroup consisting of a) the Fc region of an immunoglobulin molecule; b)the hinge region of an immunoglobulin molecule; c) the CH1 region of animmunoglobulin molecule; d) the CH2 region of an immunoglobulinmolecule; e) the CH3 region of an immunoglobulin molecule; f) the CH4region of an immunoglobulin molecule; g) the light chain of animmunoglobulin molecule; h) a peptide comprising a leucine zipper motif;and wherein said oligomerization domain is capable of forming anoligomer which is at least a dimer with at least one other polypeptidecomprising said oligomerization domain.
 30. The method of claim 29,wherein said polynucleotide has the sequence selected from the groupconsisting of: a) the nucleotide sequence set forth in SEQ ID NO:1, 3,5, 7, 9, or 11; b) a nucleotide sequence having at least 75% identity tothe nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, or 11; andc) a nucleotide sequence having at least 95% identity to the nucleotidesequence set forth in SEQ ID NO:1, 3, 5, 7, 9, or 11.